The Study On The Effect Of TanReQing Injection And Mechanismon Methicillin-resistant Staphylococcus Aureus

Objective:Since 1961,the first Methicillin-resistant Staphylococcus aureu(MRSA) was isolated,thenmany hospitals reported infections of MRSA, when in the 1980, MRSA infection was almost all over the world, It has been an important pathogen of nosocomial infections in nowdays, MRSA,hepatitis B along with AIDS,have been the world's three major problems,which are difficult to cure. Moreover the situation of drug resistance is grim, MRSA can produce resistance to many antibiotics, such as?-caprolactam, sulfonamide, Aminoglycoside, rifampicin, macrolide,fluoroquinolones, tetracycLzd. Therefore, the antibiotic which is used for MRSA is extremely limited, Vancomycin isone of the antibiotics which can kill MRSA, but in recent years the emergence of drug-resistant strains, and their own toxic side effects,limite their clinical use. Although recent years have developed some new anti-MRSA drugs, but MRSA wouldalso get drug resistance quickly. Therefore, relying solely on the development of new antibiotics can not curb the process of bacterial resistance.Traditional Chinese medicine were used to treat infectious diseases for thoustands of year andplayed important role on public health. There are a lot of qingre detoxification prescription, whichhas good antibacterial effect .So far it did not have research found the bacreria is able to produce drug-resistant to Traditional Chinese medicine.According to the results of our early work, we found that Traditional Chinese medicine are different from antibacterial drugs, some traditional Chinese medicine could damage and inhibite the biofilm at the formation and mature stage.it could also have an inhibition and removal effect to drug-resistant bacteria, which is undoubtedly different from traditional Western medicine antibiotics antibacterial advantage and highlight.In order to explore the mechanism of antimicrobial resistance of Chinese herbs,the research used Tanreqing injection (TRQ)as experimential drug to study the destructive effect on MRSA biofilm and to find the main active components, to observe the synergistic effect on MRSA with antibiotics, to investigate the effect of TRQ on resistance reversal of MRSA.Method1. Biomass determination of forming MRSA biofilmIn order to investigate the effect of TRQ on the formation of MRS A biofilm, the mixture of drug and bacterialsuspension, TRQ (serial concentrations from 16,500 to 9 mg·L-1) and Vancomycin (serial concentrations from 20 to 0.039 mg·L-1) were addedinto microplate and incubated overnightat 37?. After incubation each plate was washed twice with 0.9%NaCl to remove planktonic cells. Finally the living cells and the totalbiomass of biofilm were determined by the XTT and theCV method.2?Biomass determination of matureMRSA biofilmIn order to investigate the effect of TRQ on the mature MRSA biofilm. The bacterialsuspension was added and incubated for 24h at 37 ?,and then added the drug for 24h. After incubation each plate was washed twice with 0.9%NaCl to remove planktonic cells. Finally the living cells and the totalbiomass of biofilm were determined by the XTT and theCV method.3. Imaging observation of MRSA biofilm withCLSM2ml MRSAdilution (OD600= 0.1) was cultured in a 35mm glass bottom Petri dish for the experiment. ThenTRQand Vancomycin were added into parallel dishes after24h. 24 h later, Use Confocal Laser Scanning Microscope (CLSM) to observe theeffect after the Syto9 was added.4. Imaging observation of MRSA biofilm with SEMthe mature MRSA biofilm was incubated sterile glass,then added the drug for 24h. Scanning Electron Microscopy (SEM) was used to find out the effect of TRQ on the bacteria in the MRSA biofilm..5.Observe the antibacterial synergy effectsby thecheckerboard dilution method .The two drugs were diluted by the checkerboard method in the microplate from two directions According to the MIC of MRSA .the the vancomycin and Linezolid were diluted from 4xMIC to 1/16xMIC whereas the TRQ from8250 to 126 mg·L-1.The bacterialsuspension was added 100 uL, incubated overnightat 37? .Then each hole added the 0.25% TTC10 uL and continued to cultivate 2 h. The occurrence of red color for bacteria growth, the results of the two drugs combined was calculated fractional inhibitory concentration index,FICI).6.reserve the drug resistance of MRSAAdding the TRQ to LB medium and bacterialsuspension which made the final concentration is 1/4TRQ, concentration and incubated overnightat 37 ? Oscillation theninoculation bacteria LB solid medium, 37 ? for 24h, and repeated for 3 times,saved at 4 ?., then to test whether the resistance of MRSA was reserved by sub-MIC concentration..Result:1. The results of Microplate model showed that TRQ had a good effect on MRSA biofilm. concentration of TRQ.The results of CLSM showed that TRQ can destruct the water channels of MRSA biofilm and the scanning electron microscope showed that TRQ induced biofilm loose and MRSA in different sizes. Thus provides evidences for TRQ can destructive the MRSA biofilm architecture and can inhibit cell division.2.1t was found that at the biofilm formation stage, Scutellaria baicalensis, Bear Gall powder, Lonicera japonica, Forsythia suspensa.could inhibite the biofilm, which could reduce the living bacteria in biofilm. When the biofilm got matured, Scutellaria baicalensis, Goral horn, Forsythia suspensa. could only reduce the total biomass, the amount of live bacteria no longer function, only Bear Gall powder can significantly reduce the total bacteria and the amount of live bacteria.3. The minimal inhibitory concentration (MIC) of TRQ injection was 1/2Drug concentration;while the MIC of Vancomycin and Linezolidwere both 5 mg·L-1(MRSA).The fractional inhibitory concentration index ( FICI) of TRQ combination with Vancomycin and Linezolid were both 1.24 h time-kill curve assay showed Vancomycin andLinezolid have bactericidal activity against MRSA with the concentration of 1 x MIC?4 x MIC used alone;while 1/2Drug concentration of TRQ.When used together under the same concentrations(1/8xMICVancomycin+1/8 TRQ;1/8×MIC Linezolid+1/8 TRQ), they all showed bactericidal activity with synergy and additional effects. ?LC24 were 2.631 and 2.628.4. The results showed that the MIC of drugs were significantly decreased,.Conclusion:TRQ can inhibit the biofilm formation and destruct mature biofilm of MRSA,so it has the anti-effect on the biofilm of MRSA. The Bear Gall powder plays an important role in the anti-biofilm in TRQ .The experimental data showthe synergestic effects with TRQ and Vancomycin/Linezolid in against MRS A. After the combination of two drugs can be prolonged drug antibacterial effect.TRQ was able to reverse the resistance of MRSA.To sum up, compared with antibiotics, TRQ plays a very unique role of killing and destroying bacterial biofilm in antibiotic-resistant bacteria, and has the potential to develop into new drug, It remained the further studyabout pharmacological activities and mechanisms.