Study On The Antibacterial Activity Of Combined Antibacterial Agents Against MRSA In Vitro

PrefaceMethicillin - resistant staphylococcus aureus ( MRSA) was one of the important hospital - acquired pathogen currently, MRSA was detected in England firstly and spreaded in the universal medical organizations . A study from America suggested that the infection caused by MRSA was about 25 percentage of co-mosocal infection in 2001. The morbidity of the infection caused by MRSA has also increased in China year after year, and MRSA was not only acquired in hospital, but also in community . MRSA was resistant to aminoglycosied, tetracy-cline,sulfonamide,rifamping. Now ,the most effective drugs are Vancomycin and Teicoplanin. The situation has been changed after the first case caused by VISA (Vancomycin intermediated Straphylococcus aureus) was reported in Japan in 1996. In June 2002, the first case caused by VRSA ( Vancomycin resistant Straphylococcus aureus) was reported in America. Glycopeptide - intermediated Straphylococcus aureus has been reported in our country. Infection caused by MRSA is regarded as one of three infectious deseases which are the most difficult to solve, and it is becoming a hot - point research in the world. Because of severe - drug — resistance of MRSA, hetro - VRSA has been emerged especially. Although the cases were few, it was concerned world wide. We investigated the antibacterial activity of Vancomycin or Teicoplanin combined respectively with Rifampin or Levofloxacin against MRSA in vitro and provided evidence for controlling infection of MRSA and using antibacterial agents reasonably.Material and MethodsMaterial and EquipmentBacterial strain 30 specimens of MRSA were collected between November 2003 and February 2006 from all kinds of infectious samples of in - patients and outpatients of No. 1 Hospital Affiliated to CMU. Antimicrobial Vancomycin ( USA Lilly);Teicoplanin ( Aventis company);Rifamping ( From Shen Yang shuangding pharmaceutical company);Levofloxacin ( No. 1 pharmaceutical factory in Beijing) . Control bacteria Straphylococcus aureus ATCC 29213 provided by Bacterial Room of No. 1 Hospital Affiliated to CMU.CultureAll strains are cultured using Muller — Hinton ( MH) culture ( Beijing aubox-ing company limited);Nutrient broth is also Muller — Hinton ( USA BBL company)Equipment VITEK2 ( French BioMerieux);Spectrotograph ( England OX-OID company);96 -holed board (Made in China)MethodsBacteria were cultured and separated by routine approaches and their species were determined by VITEK, the Microbiological Automatic Analyzer. MRSA was identified through determining MIC of Oxacillin by Microdilution Method according to the criteria of American NCCLS. Broth Microdilution was applied for drug sensitive test. Vancomycin and Teicoplanin were diluted with 2ml of sterilized MH broth to 11 different concentrations;64,32???,?? -0.063jxg / ml. Rifampin and Levofloxacin were diluted with 2 ml of the same MH broth to 11 different concentrations;512, 256,......,0. 5|xg / ml. Before we used check-board method, MIC of those 4 kinds of agents used alone against subject bacteria should have been detected at first. Examination of MIC of single agent against MRSA;1. Added lOOul of suspension which concentration was 1 x 106CFU/mltoCFU/mlto each hole. 2. Added lOOul of antimicrobial. 3. Cultured in incubator under 35T! over night. 4. Investigated after 18 to 24 hours. Combined drug sensitive tests( designed according to checkboard method) : 1. Added lOOul suspension which concentration of 1 x 10 CFU/mlto each hole. 2. Added lOOul of combination of two kinds of antimicrobial randomly (50ul of each kind) to every hole. 3. Cultured in incubator under 35Tl over night. 4. Investigated after 18 to 24 hours. Recorded MICa - alone and MICb - alone when drugs were used a-lone and the MICa - combined, MICb - combined. Then caculated FIC index, (fractional inhibitory concentration index).ResultsThe MICcfl of Vancomycin against MRSA ( n = 30) was 1 jxg / ml while it was 4jxg / ml of Teicoplanin. MIC^ dropped to 0. 5jxg / ml when Vancomycin was combined with Rifampin or Levofloxacin respectively. But when it came to Teicoplanin, it dropped to 2|m,g / ml.DisccusionVancomycin and Teicoplanin are glycopeptide antibactic, and are mainly used in the therapy of gram - positive bacterial infection, which still has antibacterial activity against multi — drug - resistant Straphylococcus aureus and en-terococcus currently. It was shown that they had the same antibacterial activity in sensitivity tests invitro and animal tests models. Rifampin can be used in the therapy of MRSA infection, but in order to prevent resistance of Rifampin, it should be combined with other agents or glycopeptide. Bacteriostatic rate of Flu-oroQuinolone against MRSA was 98. 1%. Now, its sensitivity to MRSA decreased because of extensive usage in our country. In my study , Bacteriostatic rates of Vancomycin and Teicoplanin against MRSA were both 100% . But it was only 30% when Rifampin was used alone against MRSA, Levofloxacin against MRSA(n =30) was drug - resistant. Antibacterial activity is increased whenVancomycin or Teicoplanin was combined with Rifampin or Levofloxacin respectively. Antibacterial activity of Rifampin or Levofloxacin was also increased significantly. There was a synergistic effect when Vancomycin or Teicoplanin was combined with Rifampin, and there was an additive and synergistic effects when Vancomycin or Teicoplanin was combined with Levofloxacin. Drug of Glycopep-tide combined with antibiotic such as Rifampin or levofloxacin, could be applied to cure clinical severe infection caused by MRSA according to the result of drug sensitive tests.ConclusionsSynergistic action was principle when Vancomycin or Teicoplanin was combined with Refampin and the antibacterial activity increased more than 2 times. Both additive and synergistic action were observed when Vanconmycin or Teicoplanin was combined with levofloxacin, No negative effects were shown. Drug of Glycopeptide combined with antibiotic such as Rifampin or levofloxacin, could be applied to cure clinical severe infection caused by MRSA according to the result of drug sensitive tests.