The Enhanced Anti-MRSA Effect Of Catechins On β-Lactams And The Study Of Its Mechanisms

Background and purpose:β-lactam is the common used antibacterial drug in clinic. For its inappropriate use,especially abuse, there are more and more β-lactam resisitant strains in hospital. In clinictrials,90-95%Staphylococcus aureas are penicillin resistant at preasent. In Asia,70-80%Staphylococcus aureas are methicillin resistant Staphylococcus aureas (MRSA), and it istroublesome to deal with. Glucopeptides antibiotics are the main drugs against MRSA, andvancomycin is called “the last resort” to deal with the MRSA infection. But vancomycinresistant Staphylococcus aureas have appeared, therefore, it is very important to search fornew effective therapeutic drugs.Many compounds among natural drugs have the ability to enhance the antibacterialeffect of antibacterial drugs, they are called as antibacterial sensitizing agents. We foundthese agents in the raw extracts of Crataegus songorica in previous experiments. Threemonomer compounds, isolated by chemical methods, showed powerful antibacterialsensitizing effect together while showed no or little antibacterial sensitizing effect alone.One monomer compound had been identified to be catechin (C) by structure analysis whileother compounds had not identified yet because of less amounts. Herein, we purchasedseven available catechins, discovered that the combination of catechin (C), epicatechingallate (ECG) and epigallocatechin (EGC)(The proportion of C, ECG and EGC was calledC2E herein) had the antibacterial sensitizing effect through activity screening in vitro.Therefore, the antibacterial sensitizing effect was investigated in vitro and in vivo. And westudied the mechanism of the antibacterial sensitizing effect on C2E according to the mainmechanisms of resistance on MRSA.Methods:1. Chose the drugs from the available catechins which could enhance the anti-MRSAWHO-2on oxacillin on the previous work basis, determined the compatability of catechinsand its compatable proportion of them with the best antibacterial sensitizing effect which C was perceived as a central role. The anti-MRSA WHO-2effects of the compatability wereobserved with β-lactams like oxacillin, ampicillin, cefazolin, cefepime, and imipenem pluscilastatin, the effects of it against25MRSA clinical strains with these β-lactams were alsoobserved.2. The survival conditions of sepsis mice model in each group infected by MSRAWHO-2was observed after treatment with oxacillin and C2E in seven days. We alsoobserved the bacterial amounts in blood of the sepsis mice model in each group after beinggiven drugs for2h and4h and the levels of cytokines TNF-α和IL-6in serum on differenttime points.3.3. The affinities of the three drugs with PBP2a had been observed by biosensortechniques and the expression situations of the main resistant gene mecA had been observedby RT-PCR. The MRSA WHO-2bacteria which were treated by the three catechins hadbeen centrifugated and suspended again by PBS, after being incubated with daunorubicinthe concentration, some of them had been taken to identified the fluorescence idensityreleased from the intracellular bodies under the fluorescence spectrophotometer and in theconfocal laser scanning microscopy method. The expression situations about common drugefflux pumps genes in Staphylococcus aureas and had been observed after being treatedwith C2E by RT-PCR and that of the efflux pump gene mepA had observed by Real timePCR techniques.Results:1.It showed no or little antibacterial sensitizing effect when C, ECG, EGC were usedwith oxacillin alone or combined with two drugs among three, but it exsited obviousantibacterial sensitizing effect when the three drugs were comined together. It was observedafter the three drugs were dealt in orthogonal test, the compatable proportion in mass ratioof C, ECG, EGC that was1:1:1was chose in the latter experiments according to the resultsfrom the former experiments. The compatability in some concentrations combined withampicillin, cefazolin, cefepime against MRSA WHO-2exhibited antibacterial sensitizingeffect, of which the FIC values were all0.38.2. The proportions of the strains amounts of which C2E exhibiting the effect ofantibacterial sensitizing among the25MRSA clinical strains were80%,76%,88%,80%,92%under the C2E total drug concentration of16μg/mL combined with oxacillin,ampicillin, cefazolin, cefepime, and imipenem plus cilastatin. 3. The survival rate in the established MRSA WHO-2sepsis mice models which weretreated in the dosage of100mg/(kg·d) of C2E with oxacillin7days was66.7%, which wasapparently different from the group of being treated with oxacillin alone(p <0.05). Thebacterial amounts of the groups which were treated by C2E in the dosage of200mg/(kg·d)and100mg/(kg·d)were less than those of the group which was treated by oxacillin alone,which was observed after being treated for2hours (p <0.05). The IL-6levels of the groupswhich were treated by C2E in some dosage were apparently deeper than those of the groupwhich was treated by oxacillin alone, which was observed after being treated for some time(p <0.05).4. There were no high affinities between the three compounds with PBP2a. Comparedwith the group of being treated by oxacillin alone, the mecA gene had not beendownregulated after being treated oxacillin combined with C2E. The stronger fluorescenceidensity from the intracellular bodies had been observed in the group after treatment of C2Ethan other groups under the fluorescence spectrophotometer and the confocal laser scanningmicroscopy. The expression of efflux pump mepA gene in the group which was treated byC2E combined with oxacillin was apparently lower than that of the group which was treatedonly by oxacillin(p <0.05), the expression of efflux pump mepA gene in the group whichwas treated only by C2E was apparently lower than that of the group which was treatedonly by oxacillin Real time PCR(p <0.05).Conclusions:1.The compatability of catechins will enhance the susceptibility of β-lactam againstMRSA WHO-2strain, the compatibility and the mass ratio with the best antibacterialsensitizing effect is C, ECG, and EGC with the mass ratio of1:1:1.2.C2E can enhance the susceptibility of β-lactam against MRSA standard strain andclinical strains.3.C2E can apparently increase the survival rate of MRSA WHO-2sepsis model mice,the mechanism may be associated with decreasing the bacterial amounts and the levels ofIL-6.4.The antibacterial sensitizing effect is not associated with binding to PBP2a ordownregulating the expression of mecA gene, but with the increased assembly of the drugs in the intracellular bodies, which may be caused by C2E downregulating the expression ofefflux pump mepA.