A Study On The Effect Of Compound Sihuang Outside Lotion Against Methicillin-resistant Staphylococcus Aureus

ObjectiveTo study the antibacterial activity,antibacterial mechanism and bacteria biofilms formation of Compound Sihuang Outside Lotion against Methicillin-resistant Staphylococcus aureus,and expect to lay the experimental foundation and further application for clinical infectious diseases.MethodsThe study was performed by three parts:(1)Study of Compound Sihuang Outside Lotion and the Chinese medicine it contains against MRSA in vitro.Observing the growth and inhibition of the subjects MRSA under the influence of Compound Sihuang Outside Lotion,rheum officinale,rhizoma coptidis,radix scutellariae,cortex phellodendri,giant knotweed and forsythia suspensa Vahl by Kirby-Bauer;testing the minimum bacteriostasis concentration(MIC)of MRSA of Compound Sihuang Outside Lotion,rheum officinale,rhizoma coptidis,radix Scutellariae,cortex phellodendri,giant knotweed and forsythia suspensa Vahl by tube dilution method,and then investigate and compare the bacteriostatic effect between the compound and single Chinese medicine.(2)Study of antibacterial mechanism about Compound Sihuang Outside Lotion against MRSA.Different concentrations of the compound were acted on MRSA bacteria liquid,and detect its change of Alkaline phosphatase(ALP)activity,peptidoglycan activity,Potassium ions leakage and Magnesium ions leakage which expected to clarify one of the antibacterial mechanism of the compound against MRSA.(3)Study of Compound Sihuang Outside Lotion' s influence on the adhesion and formation of bacteria biofilms of MRSA.MRSA strains which had the ability to form bacteria biofilms were screened via the Congo Red Agar test(CRA).Reconstruct the model of bacterial adhesion and bacteria biofilm of MRSA in vitro.Using the microporosity plate method and CCK 8 kit to estimate the influence on bacterial adhesion and bacteria biofilm formation of MRSA when different concentration of Compound Sihuang Outside Lotion were put to use.Observating formation conditions of bacteria biofilm of MRSA under the oil immersion lens.Result(1)Compound Sihuang Outside Lotion and the single Chinese medicine it contains both can inhibit the growth of MRSA On the nutrient agar medium in different degree.The bacteriostatic circle diameter from big to small arranges in the order when in the same drug concentration:rhizoma coptidis>the compound>Scutellaria baicalensis georgi>rheum officinale>fructus forsythiae>cortex phellodendri>giant knotweed.The bacteriostatic activity dependens on the concentration of drugs,while the lower drug concentration,the smaller the bacteriostatic circle diameter.The MIC of Rhizoma coptidis,Scutellaria baicalensis georgi,fructus forsythiae and Compound Sihuang Outside Lotion againt MRSA bacteria liquid all are 31.3mg/mL,while cortex phellodendri low to 15.7 mg/mL,rheum officinale is 62.5 mg/mL,and giant knot weed bad to 125.0 mg/ml.(2)Compound Sihuang Outside Lotion can lead to the change of ALP activity,potassium ion leakage and magnesium ion leakage of MRSA bacterium suspension,but it didn' t make obvious change to the activity of peptidogly,therefore one of the compound' s antibacterial mechanisms may be related to the increasing permeability of cell wall and cell membrance.(3)34 clinical Staphylococcus aureus(S.aureus)strains are isolated in Congo Red Agar test,in which 12 strains can form bacteria biofilms accounted for 35.2%of all the strains in test.Among them the Methicillin-sensitive Staphycoccus aureus(MSSA)strains which can form bacteria biofilms account for 22.7%while the MRSA strains account for 63.6%,and the rate of forming bacteria biofilms of MRSA is higher than that of MSSA significantly(p<0.05);The strains form bacteria biofilms(PIA positive)behave high resistance to a variety of antimicrobial agents which the resistant rate of erythromycin,levofloxacin,tetracycline are 75%,50%and 66.7%respectively.The resistance rate to erythromycin and levofloxacin of the staphylococcus aureus PIA positive is higher than the staphylococcus aureus PIA negative significantly according to the results of statistics(p<0.05)and the resistant strains to vancomycin haven't been discovered.In the experiment of bacteria biofilm,Compound Sihuang Outside Lotion also has inhibitory effect on adhesion of MRSA which is was inhibited significantly compared with the negative control group(P<0.05).And furthermore,the inhibition depends on the concentration of drugs,but within a short time(3 h)all groups of drugs drug show no difference in the adhesion of MRSA after a statistical test(p>0.05).Compound Sihuang Outside Lotion can inhibit formation of bacteria biofilms in 24 h which act also depends on the concentration of drugs,and the biofilm inhibition rate of the 31.25 mL group can reach to 75.3%.In addition,excepting the 7.81 mg/L and 3.90 mg/L group,the rest of the group compared with with negative control group(RPMI1640 group)are statistically significant(P<0.05).Compound Sihuang Outside Lotion produces an effect on polysaccharides secretion extracellular of MRSA smeared with oil microscopy and the effect leads to inhibition for the formation of bacteria biofilms of MRSA.ConclusionCompound Sihuang Outside Lotion can inhibit the growth of MRSA in vitro,and one of its antibacterial mechanism may be related to the change of the permeability of MRSA bacterial cell wall and cell membrane,and it is confirmed that the Compound Sihuang Outside Lotion can affect the adhesion and formation of biofilms of MRSA.