Study On Levofloxacin-induced Effects On Biofilm Of Pseudomonas Aeruginosa And The Means Of Medicine Treatment

Pseudomonas aeruginosa is an important sources of nosocomial infection caused by pathogenic bacteria. In all the mechanisms of drug-resistant, biofilm formation in Pseudomonas aeruginosa is not only a major resistance mechanisms, but also very important reason for Treatment failure in clinic. In study of forming mechanism about biofilm of Pseudomonas aeruginosa, the forming process about biofilm of Pseudomonas aeruginosa has QS systems in addition, there is also another adjustment mechanism:PQS system. Quinolone signal molecules is the main signal molecule as a molecule of PQS control system in the biofilm forming mechanism, the structure of Quinolones is similar to the structure of quinolone signal molecule, both of them are based on quinoline ring as the main structure.What roles does Quinolones play in the formation of biofilm of Pseudomonas aeruginosa? Whether can it induce the formation of biofilms? What medicines do they have to penetrate the biofilm in clinical which can treat infections caused by biofilms of Pseudomonas aeruginosa more effective?Due to the problems above, we doing the following research.1.Levofloxacin-induced effects on biofilm of Pseudomonas aeruginosa and the effects on biofilm by other medicine.Purpose:Induce standard strains of Pseudomonas aeruginosa and wild in vitro using Levofloxacin, Observe the effect of Levofloxacin in the forming biofilm of Pseudomonas aeruginosa. Add Azithromycin, Imipenem and Ambroxol in the experiment, Observe effects of them to the formation of biofilms, Observe whether they can destroy the structure of biofilm or delay the Forming time of biofilm.Strain Source:3 Strains of Pseudomonas aeruginosa, the Standard strain ATCC27853(H007) was preserved by our laboratory, the other two wild strains is isolated from the clinical.Methods:(1)The effects on biofilm of Pseudomonas aeruginosa by Levofloxacin-induced. Use levofloxacin the concentrations of which is MIC to induce Pseudomonas aeruginosa, we obsere the Results in two way-Silver staining and Scanning electron microscope(SEM) after 40 generations-induced by using Levofloxacin, compare to the strain before Levofloxacin-induced, Observe the Levofloxacin-induced effects on biofilm of Pseudomonas aeruginosa.(2) The effects of Azithromycin, Imipenem and Ambroxol on biofilm.Use the 6 strains of Pseudomonas aeruginosa(3 was Levofloxacin-induced and the other 3 was the strains before Levofloxacin-induced) to engage the experiments which were put into the culture medium with the directly dosing(the medicine is Azithromycin, Imipenem and Ambroxol).Take another 6 same strains which were put into the culture medium with cultivating Sufficient time,let the Biofilm form. Add Azithromycin, Imipenem and Ambroxol into the one which were put into the culture medium with the directly dosing.Observe the formation of biofilm at same time. Add Azithromycin, Imipenem and Ambroxol into the one which were put into the culture medium with cultivating,use the means of oscillation and high temperature with high pressure, Observe the changes about biofilm which was formed in the environment of different medicine.Use the 6 strains of Pseudomonas aeruginosa(3 was Levofloxacin-induced and the other 3 was the strains before Levofloxacin-induced, which were put into the culture medium with cultivating Sufficient time,let the Biofilm form, Add Ambroxol into the culture medium of Pseudomonas aeruginosa, Observe what was the differences between them.Results:(1) Standard strain number H007,Wild strain numbers P012, P033,the strain after 40 generations Levofloxacin-induced number H031,H038,H039.We can found obvious difference of biofilm between Levofloxacin-induced strain and Levofloxacin-noinduced strain which were formed at same time by using the means of Silver staining and SEM.(2) From the experimental results by adding azithromycin or imipenem we can see that the forming time of biofilm with Levofloxacin-induced which added azithromycin, imipenem is more earlier than the forming time of biofilm with Levofloxacin-noinduced, the forming time of biofilm with adding azithromycin or imipenem is more earlier than that without adding azithromycin and imipenem one.The speed which biofilm recover with adding azithromycin or imipenem is more slowly than that of the one without adding azithromycin or imipenem.Biofilms Levofloxacin-induced are still intact after high temperature125℃and high pressure last 20min,but the biofilms added imipenem was gone.The biofilm of Pseudomonas aeruginosa made lots of holes in the surface both Levofloxacin-induced one and Levofloxacin-noinduced one.2. The establishment experimental model of device which biofilm formation of Pseudomonas aeruginosa in vitroPurpose:Improve the experimental model of device according to literature reported methods, use 0.22μm Millipore which biofilm grow on it to establish a experimental model of device in vitro,and use this device to Screen medicine which can through the biofilm,use HPLC to detect the concentration after the medicine through the biofilm.Strain Source:2 Strains of Pseudomonas aeruginosa, Standard strain ATCC27853 and the strain number H031 which induced by using Levofloxacin from Standard strain ATCC27853.Methods:Establish the device, repeat the experiments which Ambroxol increase the concentration of Ciprofloxacin which Penetrate the biofilm to test whether the device is created successfully.Use precision experiments to test the repeatability of the experiment.Results:In Pseudomonas aeruginosa with Levofloxacin-noinduced and without adding Ambroxol,the concentration of Ciprofloxacin is 3.136μg·ml-1 and 3.270μg·ml-1,the transmittance is 3.136 and 3.270. In Pseudomonas aeruginosa with Levofloxacin-noinduced and with adding Ambroxol,the concentration of Ciprofloxacin is 3.911μg·ml-1 and 4.3796μg·ml-1,the transmittance is 3.911 and 4.3796. In Pseudomonas aeruginosa with Levofloxacin-induced and without adding Ambroxol,the concentration of Ciprofloxacin is 1.6557μg·ml-1 and 1.6825μg·ml-1,the transmittance is 1.6557 and 1.6825. In Pseudomonas aeruginosa with Levofloxacin-induced and with adding Ambroxol,the concentration of Ciprofloxacin is 3.4660μg·ml-1 and 3.4206μg·ml-1,the transmittance is 3.4660 and 3.4206.3.The effect of penetration to use Ambroxol combining with four kinds of antibiotics on biofilm of Pseudomonas aeruginosaPurpose:Screen the antibiotic which can penetrate biofilm of Pseudomonas aeruginosa. Provid laboratory support for treating biofilm infection.Strain Source:3 Strains of Pseudomonas aeruginosa,the Standard strain ATCC27853(H007), the other are H031and H039 which were Levofloxacin-induced strain.Methods:Use Ambroxol combining with Thailand, cefoperazone/sulbactam, piperacillin/tazobactam, ceftazidime respectively for the medicine to penetrate the biofilm of Pseudomonas aeruginosa, Use these medicine as controls respectively, Use conditions of HPLC are 5%-100%MeOH/H2O,40min, Shimadzu HPLC-20A, detector with DAD, Workstation software of LC-Solution.Results:Cefoperazone/Sulbactam has peak,and the UV of peak is similar to the pure in this position, it means that Cefoperazone/Sulbactam penetrate the biofilm of Pseudomonas aeruginosa, and the quantity which penetrate the biofilm of Pseudomonas aeruginosa become larger.4. Verification experimentPurpose:Use different HPLC,In different conditions (Shimadzu LC-10A), detect the quantity of Cefoperazone/Sulbactam to verify the effect that Ambroxol combining with Cefoperazone/Sulbactam.Strain Source:2 Strains of Pseudomonas aeruginosa, Standard strain ATCC27853 and the strain number H031 which induced by using Levofloxacin from Standard strain ATCC27853.Methods:Use cefoperazone/sulbactam and cefoperazone/sulbactam+Ambroxol as the medicine through the biofilm respectively, detect the height of the peak with HPLC(Shimadzu LC-10A), UV detector is SPD-10AD, Workstation software of Zhejiang University N2000.Results:In the devices add cefoperazone/sulbactam only, the height of peak is 185.091. In the devices add cefoperazone/sulbactam+Ambroxol, the height of peak is 207.233.Research above have shown that1.Levofloxacin can induce the formation of biofilm.2.Azithromycin, imipenem has the effect to inhibit the growth of biofilm, However, it is useless to the biofilm which is grown. Ambroxol can dig holes on the surface of biofilm.3.The experiment which Ambroxol accelerates the Penetration of Ciprofloxacin is repeat successfully. 4.Cefoperazone/Sulbactam can penetrate biofilm of Pseudomonas aeruginosa, after adding Ambroxol, the concentration of Cefoperazone/Sulbactam is larger. This experiment shown that this two medicine can use together to treat the infection by biofilm of Pseudomonas aeruginosa in clinic.