In Vitro Antibacterial Activity Of Constituent CP Of Crataegus Pinnatifida Combined With β-Lactam Antibiotics Against Methicillin-resistant Staphylococcus Aureus

BackgroundMethicillin-resistant Staphylococcus aureus (MRSA) is the very important pathogen in nosocomial infection,which is one common cause of extensive deep burns and severe trauma infections.Nosocomial infection by MRSA, among Staphylococcus aureus, become more and more popular, especially in intensive care units and burn wards.The detective rates of MRSA show a obvious increase, up to above 90%.This issue brings a great challenge on the clinical treatmant and nursing.Actually, the senile,severe,immunological-deficient,neonatal,are the high-risk groups with MRSA infection.Such iatrogenic factors as long administration of antibiotics,invading manipulation and sterilisation and isolation, can result in the growing up of MRSA infection.The mechanism of MRSA resistance is mainly associated with the abundant PBP2a, a special penicillin binding protein that encoded by mecA gene.PBP2a is composed of 668 amino acids,and its molecular weight is 76.1 kDa. PBP2a has low affinity to allβ-lactam antibiotics.The soluble PBP2a (644 amino acids,74 kDa) has two domains.One is an N-terminal lobe, a centralized non-penicillin binding domain of unknown function,the other is a C-terminal transpeptidase domain.Of particular interest is the C-terminal transpeptidase domain, which has a folding pattern that is typical of the PBP transpeptidases and the serineβ-lactamases.PBP2a provides complementary transpeptidase activity for the cells in order that cell walls synthesize although other PBPs are inhibited byβ-lactams,thus cause drug resistance.Therfore, it is very important and essential to discover new antibiotics or drugs to control MRSA infection or decrease resistance of MRSA.ObjectiveThis study aims to major antibiotic-resistent protein PBP2a from MRSA as a target and to screen Chinese herb medicines that can coordinate withβ-Lactam antibiotics,which could be purified and analyzed so as to find bioactive chemicals.Methods1.Use biosensors to screen Chinese herbs that can bind to the PBP2a from MRSA.2.In vitro antibacterial susceptibility testing studies were performed to further functionally screen Chinese herbs that can enhance the antibiotic effect of Oxacillin.3.For the herbs we screened out, purify each constituent and find the most effective part that can coordinate with Oxacillin.4.Through in vitro antibacterial susceptibility testing, further evaluate the antibiotic activity of a combination of active constituent in herbs andβ-Lactam antibiotics.Result1.Among the raw extracts of 78 Chinese herbs,we found that tripterygium wilfordii hook,Crataegus pinnatifida,houttuynia cordata,radix paeoniae rubra,Cacumen PLatycladi,Rhizoma Coptidis,Fructus Terminaliae Tomentellae Immaturus,pericarpium granati and Radix et Rhizoma Rhei Palmati can bind to the C terminal of PBP2a with relatively higher affinity than 200RU.2.In vitro Antibacterial susceptibility testing demonstrated that among the 78 Chinese herbs,Crataegus pinnatifida,pericarpium granati,Flos Syzygii Aromatici,Catechu displayed combinational antibiotic activity withβ-Lactam antibiotics.Considering the binding value of Crataegus pinnatifida is 365.22RU,the highest of them.We selected Crataegus pinnatifida as the object of our research.3.Treat the decore slice of Crataegus pinnatifida with 75% alcohol and puried the active constituent with extraction,macroporous resin,silica gel.Then find the most effective CP consituent for combined antibiotic activity.4.Consituent from CP can significantly enhance the antibiotic activity of oxacillin,ampicillin sodium-sulbactam sodium,ampicillin,cefazolin,decrease the MIC of manyβ-Lactam antibiotics,and increase the sensitivity of MRSA toβ-Lactam antibiotics.ConclusionThis study used biosensors and in vitro antibacterial susceptibility sensitive test to screen Chinese herbs and found that Crataegus pinnatifida can have combinational antibiotic activity with Oxacillin against MRSA.Active constituents from Crataegus pinnatifida were then extracted and analyzed by in vitro combination medicine sensitive test, and then the most effective part for combinational antibiotic activity withβ-Lactam antibiotics was obtained.Through in vitro medicine sensitive test of highly antibiotic resistent MRSA lines,we cofirmed that the active constituent CP can effectively lower the minimum inhibitory concentration ofβ-Lactam antibiotics and enhance the sensitivity of MRSA, then identified Catechin from the constituent of CP, which provide a new clue for clinical treatment of antibiotic resistent MRSA infection.