以水凝胶为基质的体外新型铜绿假单胞菌生物被膜模型的建立  

Creation of a novel hydrogel-based model of Pseudomonas aeruginosa biofilm in vitro

作　　者：周林颖 佘鹏飞 陈丽华[1] 罗振[1] 廖金凤 伍勇[1] ZHOU Lin-ying;SHE Peng-fei;CHEN Li-hua;LUO Zhen;LIAO Jin-feng;WU Yong(Clinical Laboratory,The Third Xiangya Hospital Affiliated with Central South University,Changsha,China 410013)

机构地区：[1]中南大学湘雅三医院检验科,湖南长沙410013

出　　处：《中国病原生物学杂志》2020年第2期125-130,135,共7页Journal of Pathogen Biology

基　　金：湖南省科卫联合基金项目(No.2019JJ80029)。

摘　　要：目的在盖玻片和导尿管上分别建立以水凝胶为基质的体外新型铜绿假单胞菌生物被膜模型模拟体内组织细菌感染情况。方法通过XTT比色法确定最适合铜绿假单胞菌成膜的热灭活马血清浓度,并运用结晶紫染色法比较不同种类的血清对铜绿假单胞菌生物被膜形成的影响。用波长为366nm的紫外线照射制备含有热灭活马血清的半固体水凝胶,以其为基质建立铜绿假单胞菌生物被膜模型。用结晶紫染色观察盖玻片上生物被膜形态结构,激光共聚焦显微镜观察生物被膜的位置分布及立体结构。用结晶紫比色法和平板稀释菌落计数法测定导尿管上生物被膜的形成量,并用扫描电镜观察细菌在水凝胶中的分布和生长状态。结果热灭活马血清浓度达到6.25%时即可促进生物被膜形成,并且热灭活马血清、热灭活人血清和胎牛血清均可促进生物被膜生长,三者之间没有统计学差异(F=0.24,P>0.05;F=0.91,P>0.05)。与液体基质构建的传统模型相比,添加6.25%热灭活马血清构建出的以水凝胶为基质的新型模型中生物被膜结构更为紧实,盖玻片经染色后光学显微镜下观察可见细菌相互聚集、粘连形成了复杂的网状结构,激光共聚焦显微镜下可见生物被膜分布密集,层叠如积云状,棉絮样,有立体层次感。此外,该模型生物被膜形成量也显著增加。结晶紫比色法测得硅胶导尿管中PAO1和PA46生物被膜形成量(A570值)分别从0.69±0.08和0.79±0.06增加至1.54±0.38 (t=3.76,P<0.01)和1.82±0.26(t=6.82,P<0.01),平板稀释菌落计数法测得PAO1和PA46生物被膜中细菌数(cfu/ml)分别从(1.06±0.16)×10^7和(1.19±0.48)×10^7增加至(2.84±1.29)×10^7(t=2.49,P<0.05)和(3.77±0.33)×10^7(t=3.62,P<0.01)。扫描电镜下可见细菌形态正常,相互聚集交融于水凝胶基质中。结论以水凝胶为基质的新型铜绿假单胞菌生物被膜结构更为紧实,成膜量也显著增加,该模型为�Objective A novel hydrogel-based model of Pseudomonas aeruginosa biofilm in vitro was created on coverslips and urinary catheters to simulate the realities of a bacterial infection in tissues in vivo.Methods The XTT assay was used to determine the concentration of heat-inactivated horse serum that was most suitable for biofilm formation,and the effect of different types of serum on biofilm formation by P.aeruginosa was evaluated by measuring the absorbance at 570 nm after crystal violet staining.A semi-solid hydrogel containing heat-inactivated horse serum was prepared using ultraviolet irradiation at a wavelength of 366 nm.Coverslips and urinary catheters were inserted into the hydrogel to create a biofilm model.The morphological structure,position,and distribution of biofilm on the coverslips was observed with an optical microscope(crystal violet staining)and a confocal laser scanning microscope.The volume of biofilm on urinary catheters was quantitatively determined using a crystal violet assay and plate dilution colony count,and the distribution and growth of the bacteria in the hydrogel was observed using electron scanning microscopy.Results Treatment with 6.25%heat-inactivated horse serum promoted biofilm formation(P<0.05),and the three types of serum(heat-inactivated horse serum,heat-inactivated human serum,and fetal bovine serum)did not differ significantly in promoting biofilm formation by PAO1 and PA46(F=0.24,P>0.05;F=0.91,P>0.05).Finally,a novel hydrogelbased model of biofilm containing 6.25%heat-inactivated horse serum was successfully constructed.Compared to the traditional model on a liquid substrate*the model created here had a compact biofilm structure.After staining,the coverslips indicated that the bacteria aggregated and adhered to each other to form a complex network structure when observed under the optical microscope.The biofilm was layered and densely distributed;cumulus-like layers were stacked when observed under the confocal laser scanning microscope.The volume of biofilm also increased

关 键 词：铜绿假单胞菌 生物被膜 水凝胶 热灭活马血清 

分 类 号：R378.991[医药卫生—病原生物学]