| [BACKGROUND] Biofilm related infection caused by Pseudomonas aeruginosa (PA) is common infection difficulty to treat in clinic. The characters of biofilm bacteria of increasing antibiotic resistance and not easy to be swallowed or lysised by WBC, are main reasons that biofilm may cause infection chronic or persistent. According to the recent data provided by NIH, more than 60% bacteria infections are related with biofilms.Therefore, study on biofilm formation and its influencing factors, mechanisms of antibiotic resistance, is important meaningful to explore new methods and new techniques for biofilm related diseases.[OBJECTIVE] (1) To screen an effective and practical method for analyzing PA biofilms; To compare the difference of PA biofilms formation in different conditions, including incubated time, temperature, attaching materials, flowing speed with selected biofilm analysis method as above screened result. (2) To determine the effectiveness of a new intervention strategy- destroying the biofilm matrix with bio-enzymes complex, to increase susceptibility of PA biofilm to ceftazidime. (3) To study the inducing ability and their difference of various β-lactam antibiotics for ampC mRNA expression in PA biofilm and to explore some resistance mechanisms of PA.[METHODS] (1) Biofilms models are established in continuous culture and static culture, respectively. We employed four methods for biofilm analysis: Confocal Laser Scanning Microscopy (CLSM), Scanning Electron Microscopy (SEM), crystal violet staining and viable bacteria counting. Biofilms that are formed in batch (static) culture are measured in absorbance with crystal violet staining. PA biofilms established in chemostat-MRD (modified Robbin device) were detected in other 3 methods. With viable bacteria counting method, comparing the biofilm formation differences in different incubation condition: various incubation time (8h,24h,72h), various temperature,variousadherent materials, various flow velocities and different medium. (2) After PA biofilms model established on silicone slides in MRD for 8h, 24h, 48h, 72h and 96h with M63 as nutrient source, the slides were immersed 3h in M63 with and without bio-enzyme complex, the mixture of enzymes (5000u streptodornosa per ml and 4000u chymotrypsin per ml) without microbicidal effect. The numbers of viable cell in biofilms of different time groups were counted, respectively. In another experiment, four groups of PA biofilms (48h) models were established with MRD, then irrigated with (i)M63, (ii)M63 with ceftazidime (23ug/ml), (iii)M63 with bio-enzyme complex for 3h at 6-hours intervals, (iv) bio-enzyme complex for 3h at 6-hours intervals basing the continuously using M63 and ceftazidime (23|ag/ml) for 24h. The numbers of viable cell in biofilms of 4 different groups were counted and synergism of bioenzyme complex to ceftazidime to kill biofilm bacteria were determined. (3) 5iofilms of PA 01 model were formatted with MRD after incubation 72h, and then exposed to some kinds of pMactam antibiotics for 7h. The bacteria cells in the biofilms were collected and total RNA were extracted and reverse transcripted to cDNA. The mRNA expression of ampC was compared by semi-quantitative PCR and quantitative PCR.[RESULTS] (1) Three-dimensional structure of biofilms and density of microcolonies in biofilm were observed in CLSM images. The surface structure of biofilms and the magnitude of irregular matrix can be observed in SEM images. The absorbance of bioflims can be detected with crystal violet staining method. The biofilms can be analyzed quantitatively with viable bacteria counting method. (2)The numbers of viable bacteria (CFU/disc) in biofilms were measured in groups of different culture time (8h, 24h, 72h), different temperature (23 °C, 37°C), different adherent materials (glass, silicone), different flow velocities (30ml/h, 90ml/h) and different culture medium (M63 medium, MH broth, LB broth). Keeping the culture temperature, medium, flow velocity and adherent material invariable, the numbers of viable bacteria (LogioCFU/disc) in 8h, 24h and 72h biofilms are 4.01±0.26, 4.59±0.49 and 5.20±0.47, respectively (PO.001). Keeping the culture time, medium, flow velocity and adherent material invariable, the numbers of viable bacteria (LogioCFU/disc) in biofilms in 23 °C and 37°C are 5.12±0.43, 5.30±0.42, respectively (P= 0.039). Keeping the culture temperature, time, medium, flow velocity invariable, the numbers of viable bacteria (LogioCFU/disc) in biofilms grown on the surfaces of glass and silicone are 5.49±0.59 and 6.2U0.40, respectively (PO.001). Keeping the culture temperature, time, medium and adherent material invariable, the numbers of viable bacteria (LogioCFU/disc) in biofilms grown in the flow velocities of 30ml/h and 90ml/h are...
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