阿奇霉素和克拉霉素对铜绿假单胞菌生物膜形成的作用

摘要

目的 研究阿奇霉素和克拉霉素对铜绿假单胞菌生物膜形成的作用.方法 微量肉汤稀释法测定阿奇霉素、克拉霉素、头孢他啶、环丙沙星和庆大霉素对4株铜绿假单胞菌的最低抑菌浓度(MIC);银染法鉴定使用医用微孔滤膜建立的铜绿假单胞菌生物膜体外模型的生物膜形成情况;结晶紫染色法检测阿奇霉素和克拉霉素对生物膜粘附性的影响;噻唑蓝法检测阿奇霉素和克拉霉素分别与头孢他啶、环丙沙星或庆大霉素的协同作用.结果 铜绿假单胞菌ATCC 27853、PAl、PA2和PA3对阿奇霉素、克拉霉素、头孢他啶、环丙沙星和庆大霉素的MIC值分别为(32,64,1,≤0.125,0.25 μg/mL)、(32,256,8,0.25,lμg/mL)、(64,128,＞256,32,2μg/mL)和(64,128,8,2,0.25μg/mL);银染法发现4株铜绿假单胞菌的培养微孔滤膜表面覆盖物均为灰黑色,浓厚致密,呈棉絮状膜样;与不加抗菌药物对照组相比,加入1/16 MIC或1/4 MIC的阿奇霉素以及1/16 MIC或1/4 MIC的克拉霉素均可显著降低4株菌生物膜的粘附性(P均＜0.05);与单独使用1 MIC头孢他啶、环丙沙星和庆大霉素相比,各抗菌药物联合1/16 MIC的阿奇霉素或1/16 MIC的克拉霉素均可分别降低以上4株菌生物膜中的活菌数量,差异有统计学意义(P均＜0.01).结论 阿奇霉素和克拉霉素可有效抑制铜绿假单胞菌生物膜的形成,并与头孢他啶、环丙沙星或庆大霉素联合应用有协同作用.%Objective To investigate the effects of azithromycin and clarithromycin on biofilm formation of Pseudomonas aeruginosa.Methods The minimal inhibitory concentrations (MIC) of azithromycin,clarithromycin,ceftazidime,ciprofloxacin and gentamicin against 4 strains of Pseudomonas aeruginosa (ATCC 27853,PA1,PA2 and PA3) were determined by broth microdilution method.The biofilm formation of Pseudomonas aeruginosa was identified by silver dyeing method on the biofilm model with medical microporous membranes in vitro.The effects of azithromycin and clarithromycin on the adhesion of biofilm were detected by crystal violet staining.The synergistic bactericidal effects of azithromycin and clarithromycin with ceftazidime,ciprofloxacin or gentamicin were detected by thiazolyl blue method respectively.Results The MIC values of Pseudomonas aeruginosa ATCC 27853,PA1,PA2 and PA3 for azithromycin,clarithromycin,cefiazidime,ciprofloxacin and gentamycin were 32,64,1,≤0.125 and 0.25 μg/mL;32,256,8,0.25 and 1 μg/mL;64,128,＞ 256,32 and 2 μg/mL and 64,128,8,2 and 0.25 μg/mL.The results of silver staining showed that the microfiltration membranes of all the four P.aeruginosa cultures appeared to be grayish black,thick and dense,and cotton-like.Compared with the control group in which no antibacterial drugs were added,both azithromycin and clarithromycin (1/16 or 1/4 MIC) reduced significantly the adhesion of the 4 strains of Pseudomonas aeruginosa (ATCC 27853,PA1,PA2 and PA3) on the microporous membrane (Pall ＜ 0.05).Compared with the use of 1 MIC of ceftazidime,ciprofloxacin or gentamycin alone,the combination of anyone of the four antimicrobial drug with azithromycin or clarithromycin (1/16 MIC) reduced the counts of viable bacteria on each biofilms with significant differences (P all ＜ 0.01).Conclusion Azithromycin and clarithromycin could effectively inhibit the biofilm formation of Pseudomonas aeruginosa and may present synergistic effects combined with cefiazidime,ciprofloxacin or gentamicin.