In Vitro Antibacterial Activity Of Daptomycin On The Multidrug-resistant Staphylococcus Aureus (MRSA)

Methicilin resistant Staphylococcus aureus(MRSA),an important bacterial strain in clinic,is resistant to the following antibiotics: ?-lactam antibiotics(including carbapenems),aminoglycoside,macrolides,clinadamcin,tetracycline and quinolone.Vancomycin is the first choice for the clinical treatment to MRSA,but intermediate-level resistant to vancomycin of Staphylococcus aureus is also found recently.As the last choice of treatment to MRSA infection,vanconmycin is facing challenges.Amphipathic cyclic lipopeptide Daptomycin(DAP),one of cyclic lipopeptide exhibits novel mechanism,whose target is the bacterial membrane.DAP kills bacteria with the help of calcium.In this study MRSA was isolated from the sputum of clinical patients.The objective is to study the antibacterial activity of DAP against MRSA in vitro and continuous action effects.The reference object is the analogue of L-K6 which was separated and purified from the skin secretion of Chinese forest frogs,while the control bacterium Staphylococcus aureus(ATCC 29213).We investigated the antibacterial activity of DAP(MIC method),and the bacteriacidal dynamics with different drug concentration contact time(Time-killing curve)and post antibiotic effect(PAE)by the way of palte count.Then we examined the effection of DAP on the permeability of bacterial inner and outer membrane.Finally,we detected the expression difference of mecA,fnbA,fnbB and sarA with RT-qPCR before and after the addition of DAP.The results showed that,in the present of calcium,DAP showed strong antibacterial effect against the selected bacteria,the MIC value is 0.78?mol/L.But in the absent of calcium,the antibacterial activity was moderate and the value of MIC is12.5?mol/L for sensitive Staphylococcus aureus,while 25?mol/L to MRSA.The results of time-killing curve and PAE showed that,with the increasing concentration of DAP,the killing time was shortened,and the killing effect was enhanced.The killing effect was obvious concentration-dependent in both bacteria.DAP produced obvious postantibiotic effect on both bacteria,and showed concentration-dependent effect.The effect of DAP on the permeability of bacterial inner and outer membrane showed that DAP had strong depolarization to inner membrane with concentration-dependent,and almost no relation to bacterial drug resistance.The effect of DAP on the permeability of bacterial outer membrane was not obvious,which might be due to the direct site of action of DAP on Staphylococcus aureus was cell inner membrane,not cell outer membrane.Results on RT-qPCR showed that the gene mecA,fnbA,fnbB and sarA were all negative in sensitive Staphylococcus aureus,while mecA,fnbA and sarA were positive in MRSA.But after DAP treatment,the expressions of mecA,fnbA and sarA were generally inhibited with concentration-dependent.