mucA基因突变的黏液型铜绿假单胞菌PA17与PD0300生物被膜形态观察和耐药性比较

BIOFILM STRUCTURE AND RESISTANCES TO ANTIBIOTICS OF MUCOID PSEUDOMONAS AERUGINOSA WITH mucA GENE MUTATION

目的研究新的mucA基因缺失突变的黏液型铜绿假单胞菌PA17和经典mucA基因点突变的黏液型铜绿假单胞菌PD0300在生物被膜状态下对临床常用抗菌药物的耐药性变化,探讨mucA基因突变对铜绿假单胞菌生物被膜形态及细菌耐药性的影响。方法改良的平板法建立生物被膜,将含绿色荧光蛋白的pUCP/UV质粒转化两株铜绿假单胞菌,激光共聚焦显微镜下观察生物被膜形态;采用琼脂二倍稀释法测定常用β-内酰胺类,氨基甙类,喹诺酮类抗菌药物对铜绿假单胞菌菌株PA17、PD0300的最低抑菌浓度(MIC);利用96孔板建立生物被膜测定抗菌药物对第五天成熟生物被膜内细菌的最低杀菌浓度(MBC)。结果新的mucA基因缺失突变的黏液型PA17成熟生物被膜呈薄膜状、经典mucA基因点突变的黏液型PD0300成熟生物被膜呈蘑菇状;在浮游状态下PA17、PD0300对头孢他啶(CAZ)、妥布霉素(TOB)、庆大霉素(GEN)、亚胺培南(IPM)敏感,而对左氧氟沙星(LVX)、环丙沙星(CIP)不敏感,两者具有一致的耐药性;生物被膜状态下两者对抗菌药物敏感性降低20-8000倍;黏液型PD0300成熟生物被膜对抗菌药物敏感性低于黏液型PA17。结论黏液型铜绿假单胞菌在相同条件下能形成不同形态的生物被膜;生物被膜状态下较浮游状态下对常用抗菌药物敏感性明显下降,同时生物被膜形态也影响抗菌药物敏感性。

Objective To research the resistance to clinically common antibiotics of mucoid Pseudomonas aeruginosa PA17 with new mucA gene deletion mutation and mucoid Pseudomonas aeruginosa PDO300 with classic mucA gene point mutation, and investgate the influence ofmucA gene mutation on biofilm structure and on bacterial resistances. Methods Transfecting pUCP/UV plasmid with green fluorescent protein into Pseudomonas aeruginosa, we used confocal laser scanning microscopy to observe biofilm formation. We separately cultured biofilms and determined the minimum inhibitory concentration （MIC） of planktonic bacterial by double agar dilution method and the minimum bactericidal concentration （MBC） of mature biofilms in 96-well plates on the fifth day to fl-lactams, aminoglycosides and quinolone antibiotics. Results Under confocal laser scanning microscopy mucus-type PA 17 slowly shaped into the flat-film feactures in the mature biofilm, but, the features of mucus-type PDO300 biofilm were like mushroom. In the planktonic state, PAl7 and PDO300 were sensitive to ceftazidime（CAZ）, tobramycin（TOB）, gentamicin（GEN）, imipenem （IPM）, but not to levofloxacin （LVX） and ciprofloxacin （CIP）. Both of them had the same resistance. However, in the mature biofilm state both of them had reduced susceptibility to antibiotics between 20-8000 times. The sensitivity of mucous type PDO300 to antibiotics was weaker than that of mucoid PA 17. Conclusion It is possible that mucoid Pseudomonas aeruginosa have different biofilm structures in the same condition. Compared with planktotic bacteria, the biofilm reduced the sensitivity to antibiotics. The biofilm structure also affects the sensitivity to antibiotics.