Staphylococcus Aureus Lux Reporters In New Antibiotic Discovery

Staphylococcus aureus is a major nosocomial pathogen,not only can it cause purulent infection of soft tissue,but also cause meningitis,endocarditis,sepsis and other serious symptoms which are life-threatening.The discovery of antibiotics is a feat in human medical history.But in recent yesrs,the abusement of antibiotics leads to the emergence of a large number of antibiotic-resistant bacteria,including methicillin-resistant Staphylococcus aureus(MRSA).MRSA is resistant to almost all kinds of antibiotics except vancomycin,and recent years even vancomycin-resistant S.aureus has been found in USA.In order to prevent the treatment of drug-resistant bacteria from falling into an untreatable state,it is necessary for us to design a new method to search for trace amount of antibiotics existed in the environment.Traditional methods of drug discovery mainly include bioactivity-oriented screening,chemical structure screening and gene cluster detection,which are difficult to develop,long cycle and difficult to screen large amounts of samples.In order to overcome these problems,it is necessary to design a reporting system with high sensitivity,high repeatability,simple operation and suitable for high throughput rapid detection of environmental samples.In this study,a series of S.aureus lux reporters were constructed:S1,S2,S3 and S4.After confirming that these reporters could emit bioluminescence when exposed to antibiotics,we use them to search for antibiotics existed in the fermentation broth and then do some work about seperation and purification.we choose seven categories of antibiotics according to their different bacteriostatic mechanisms to detect the response of reporter strains.Bioluminescence was successfully captured around the bacteriostatic zone with the help of in vivo imaging system(IVIS).Ciprofloxacin,gatifloxacin,daptomycin and mitomycin C can cause intense luminescence of four reporter strains,and different reporter strains response differently to different kinds of antibiotics.Due to the tedious process of disk diffusion test,we need a more convenient method to detect antibiotics.In this paper,we put reporter strains into 96-well plates,then fermentation samples were added into the liquid,and bioluminescence was detected by microplate reader.Three samples were finally picked from 118 samples because they can raise the bioluminescence intensity of reporter strains.Sample of No.63,which came from the fermentation broth of Micromonospora A29/GC15-SARP,a rare actinomycete,has the most obvious bacteriostasis.Subsequently,the antibiotics was isolated by gel filtration chromatography and reverse phase chromatography,and finally located at a single peak on HPLC.Due to the low yield of this antibiotic,we explored the fermentation conditions and found that this strain could produce a larger antimicrobial circle by fermentation with LB medium.