【作者】 李钊；

【导师】 蒋捍东；

【作者基本信息】 青岛大学 ， 内科学， 2008， 硕士

【摘要】 目的:研究铜绿假单胞菌在不同培养基中对不同抗菌药物的敏感性,并分析生物膜形成对β—内酰胺酶产生的影响。方法:分别选取产超广谱β-内酰胺酶、头孢霉素酶和金属型β-内酰胺酶的铜绿假单胞菌菌株,在三种培养基中建立铜绿假单胞菌生物膜模型;选用临床抗铜绿假单胞菌药物,分别在生物膜形成前后测定最低抑菌浓度(MIC)和最低生物膜菌清除浓度(MBEC)。建立产TEM、CARB酶的铜绿假单胞菌生物膜,用低剂量抗菌药物进行诱导后,采用实时定量PCR方法检测β—内酰胺酶的表达水平。结果:在浮游状态下,同株铜绿假单胞菌在三种培养基中对同种药物的敏感性无统计学差异(P＞0.05);在生物膜状态下,铜绿假单胞菌可耐受≥64倍于浮游菌MIC的头孢他啶,16～128倍于浮游菌MIC的亚胺培南,4～32倍于浮游菌MIC的环丙沙星;铜绿假单胞菌形成生物膜后,三种培养基对细菌对敏感性的影响存在统计学差异(P＜0.05)。铜绿假单胞浮游菌、生物膜菌在诱导前后都有β—内酰胺酶的表达;诱导前及经CIP、CAZ和IMP诱导后的铜绿假单胞生物膜菌所产TEM酶分别为诱导前浮游菌的1.77、13.85、16.37、34.58倍;所产CARB酶分别为诱导前浮游菌的2.94、9.53、10.84、21.90倍。诱导后浮游菌所产TEM酶的增加幅度分别为诱导前的2.04、3.31、8.65倍,而诱导后生物膜菌为诱导前的7.82、9.25、19.54倍;诱导后浮游菌所产CARB酶分别为诱导前的3.51、5.88、8.77倍,而诱导后生物膜菌为诱导前的3.24、3.68、7.45倍。结论:不同环境可以影响生物膜状态下铜绿假单胞菌对抗菌药物的敏感性。生物膜可以影响铜绿假单胞菌产生不同β—内酰胺酶的能力;铜绿假单胞生物膜菌经诱导后产TEM酶的能力强于产CARB酶。更多还原

【Abstract】 Objective: To investigate susceptibility of Pseudomonas aeruginosa biofilm strains generating different types ofβ-lactamase to antibacterials in different culture media. And investigate the effect of Pseudomonas aeruginosa biofilms on different genotypesβ-lactamases.Methods: Select P. aeruginosa isolates generateing expanded spectrumβ-lactamases （ESBL）, Cephalosporinaseβ-lactamase （AmpC） or Metalloβ-lactamases （MBL）, and set up the biofilm moulds in three kinds of media: L-Broth（LB）, Tryptic Soy Broth（TSB） and Mueller-Hinton Broth（MHB）. Biofilms were established using a modification of the Calgary biofilm device. This technique involved the generation of biofilms on plastic pegs attached to the lids of 96-well microtiter plates; Determine minimal inhibiting concerntration（MIC） and minimal biofilm elimination concerntration （MBEC） to three kinds of antibacterials, which are typically used against Pseudomonas aeruginosain cilinic: Ciprofloxacin. Ceftazidime and Imipenem, with micro-dilution and MBEC^TM-Device method. The clinical Pseudomonas aeruginosa strains known genotype of beta-lactamase of TEM and CARB were collected and induced by three antimictobials: Ciprofloxacin、Ceftazidime and Imipenem to generate beta-lactamases after the biofilms were built. And the differet expression of beta-lactamases were determined in above status by polymerase chain reaction （PCR）. The results were statistical analysized with t test and U test.Results: There was no difference in the susceptibility for planktonic strains to antimicrobials, in LB, MHB and TSB media in statistics （P >0.05）; While after the formation of biofilms,the biofilm strains could be resistant above 64 times MIC of planktonic strains to Ceftazidime , 16～128 times MIC of planktonic strains to Imipenem, and 4～32 times MIC of planktonic strains to Ciprofloxacin, respectively. And after the formation of bacterial biofilms. there existed different effects of culture media on bacterial susceptibility to Ciprofloxacin and Imipenem different media in statistics: LB<MHB<TSB （P < 0.01） ; The noninduced biofilm cells and induced by CIP、CAZ and IMP all produced much more beta-lactamases, and the quantity of TEM-beta-lactamase were 1.77, 13.85, 16.37, 34.58 times more than that of noninduced planktonic cells, and CARB-beta-lactamase were 2.94、9.53、10.84、21.90 times more than that of noninduced planktonic cells, respectively. The amplitude of TEM beta-lactamase produced by induced planktonic cells were 2.04、3.31、8.65 times more than that of noninduced planktonic, and the amplitude of TEM-beta-lactamase produced by induced biofilm cells were 7.82、9.25、19.54 times more than that of noninduced planktonic cells. The amplitude of CARB-beta-lactamase produced by induced planktonic cells were 3.51、5.88、8.77 times more than that of noninduced planktonic cells, and the amplitude of CARB-beta-lactamase produced by induced biofilm cells were 3.24、3.68、7.45 times more than that of noninduced planktonic cells.Conclusion: Different microenvironments cannot influence the susceptive of Pseudomonas aeruginosa planktonic cells to antimicrobials, but influence biofilm cells. The formation of biofilm could influence the ability of Pseudomonas aeruginosa strains to produce different beta-lactamase; and the ability to produce TEM-beta-lactamase was stronger than CARB-beta-lactamase.更多还原