Screening Of Strains For Methicillin-resistant Staphylococcus Aureus And Studies On Its Material Basis

Methicillin-resistant Staphylococcus aureus(MRSA)is a kind of hospital infection caused by multi-resistant bacteria,with strong toxicity,resistance,and easy to spread widely,etc.MRSA infection has been recognized as one of the most difficult infectious diseases in the world.At present,there are still few effective therapeutic drugs available in clinical practice.Screening of candidate compounds for antagonising MRSA is important to develop the drug treatment of MRSA infection.This study is based on finding strains that can inhibit MRSA significantly.Active pharmaceutical ingredients that against MRSA were studied on oriented extraction,isolation,purification,stability research,safety evaluation and content determination,the resrarch achievements were as follows:1.5 of 246 strains showed antibacterial activity.Further screening to determine to 1012-4 strains which showed the strongest activity for inhibit MRSA as research strain,The morphology observation,physiological,biochemical identification with API50 CHB and 16s rDNA sequence analysis were carried out to determine the strain type,as a result,it was identified as Bacillus subtilis.2.The optimal composition of culture medium produced by strains 1012-4 was glucose 20g/L,dry yeast 5g/L,MgSO4 0.5 g/L;the optimal seed fermentation conditions was culture temperature 37?,inoculation quantity 3%(V/N),with fluid volume 200mL/500mL bottle,cultivate 24h;and optimal fermentation conditions of fermentation tank is glucose:20g/L,yeast anointed 5g/L,MgSO4 0.5 g/L,culture temperature 37?,cultivate inoculation quantity 3%(V/N),rotation speed 150rpm/min,tank pressure 0.06 MPa,minute respiratory volume 30L.3.The optimal macroporous adsorption resin for extracted active substance was NKA-2.The main technical parameters as follows:adsorption velocity was 2BV/h,adsorption volume was 5%(5g resin can adsorb100mL fermented broth),washing volume was 6BV,desorption velocity was 1BV/h,the 15%ethanol as eluent solvent.By comparison of activity,the inhibitory activity of eluent is stronger than vancomycin and norvancomycin4.First discovered compound M produced from the strain 1012-4 has strong antibacterial activity to MRSA which is much stronger than vancomycin and norvancomycin;sums up the separation and purification technology of the active compound M by high performance liquid chromatography(HPLC)method.The chromatography conditions as follows:chromatographic column:Boston Crest ODS(250 mm×21.2 mm,5?m),detected wavelength:208nm,mobile phase:methanol-water,gradient elution,velocity:8mL · min-1.After repeated purification,the purity of compound M can be up to more than 98%.5.Preliminary stability test was carried out to reveal the stability of compound M,the results show that the compound M has good stability for temperature;and has a good stability between pH4.0 and pH9.0,sensitivity to pepsin and trypsin is not;the acute toxicity tests suggest that compound M has good security,under the as high as 400 mg/kg dose,the mice were no exception.6.Established the reversed phase high performance liquid chromatography(HPLC)method to determine the content of compound M in fermented liquid.The condition is:chromatographic column:Boston Crest ODS(250 mm×4.6 mm,5 ?m);detected wavelength:208nm;Mobile phase:methanol-water(3:97);Velocity:1mL · min-1;Column temperature:room temperature.The results showed that a good linear relationship was established in the range 1.0?g?8.0?g for compound M with the regression equation Y=908582X+345052,(r=0.9998).This method was simple,reliable,and suitable for content determination and quality control of compound M.