铜绿假单胞菌密度感应系统对铜绿假单胞菌-烟曲霉菌早期混合生物被膜的体外作用研究

Study on the in vitro effect of Pseudomonas aeruginosa quorum sensing system on early polymicrobial biofilm of P. aeruginosa-Aspergillus fumigatus

目的探讨铜绿假单胞菌密度感应系统(QS)对早期铜绿假单胞菌-烟曲霉菌混合生物被膜(BF)的体外影响。为临床相关混合微生物感染的治疗提供依据。方法体外建立野生型铜绿假单胞菌(PAO1)和铜绿假单胞菌密度感应缺陷菌株(P.aΔlasI/rhlI)分别与临床分离的烟曲霉菌株(A.f)形成混合BF,及单独野生型铜绿假单胞菌(PAO1)、铜绿假单胞菌密度感应缺陷菌株(P.aΔlasI/rhlI)、烟曲霉菌生物被膜,pH为7.0的RPMI-1640组作为空白对照组,于孵育24h,用结晶紫染色法半定量检测生物被膜形成能力;二甲氧唑黃(XTT)比色法评价生物被膜体外生长动力学;正置电子显微镜及扫描电镜观察生物被膜表面形态。结果结晶紫染色法半定量检测24h生物被膜形成能力提示,P.aΔlasI/rhlI+A.f混合生物被膜厚度大于其他各组(P<0.05);PAO1+A.f混合生物被膜厚度与单纯烟曲霉生物被膜组比较差异无统计学意义;XTT比色法生长动力学检测显示,P.aΔlasI/rhlI+A.f混合组活力强于PAO1+A.f组(P<0.05);正置电子显微镜及扫描电镜观察生物被膜表面形态提示,镜下P.aΔlasI/rhlI+A.f混合生物被膜中可见烟曲霉菌丝密度大于PAO1+A.f组,PAO1+A.f组菌丝密度较单独A.f组减少。结论 QS对野生型铜绿假单胞菌(PAO1)+烟曲霉菌株(A.f)早期混合生物被膜的形成起抑制作用。

OBJECTIVE The in vitro effect of Pseudomonas aeruginosa quorum sensing system (QS) on early polymicrobial biofilm of P aeruginosa - Aspergillus fumigatus (A.f) was investigated , in order to provide the basis for the treatment of clinical related polymicrobial microorganism infection. METHODS The wild-type P aeruginosa (PAO1)-clinical A fumigatus strain and the P aeruginosa quorum sensing defective strain (P.a △lasl/rhlI)- clinical A fumigatus strain were established in vitro and mixed to form the polymicrobial biofilms. Biofilms of solely the wild-type P aeruginosa (PAO1), P aeruginosa quorum sensing defective strain(P.a ΔlasI/rhlI) or A fumigatus were also developed. The RPMI-1640 group with pH 7 was used as a blank control. After incubated for 24 h, the biofilm formation ability was semi-quantitatively detected by crystal violet staining. The growth kinetics of biofilm in vitro was evaluated by 2,3-Bis-( 2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide (XTT) colorimetric method, and the ultrastructure of biofilm was observed under positive electron microscope and scanning electron microscope. RESULTS Crystal violet staining and semi-quantitative detection of 24h biofilm formation showed that the thickness of polymicrobial biofilm formed by the group of P aeruginosa quorum sensing defective strain (P.a ΔlasI/rhlI)+ A fumigatus was higher than those of the other groups (P<0.05), whereas there was no significant difference between the thickness of biofilms of the PAO1 +A.f group and the A fumigatus alone group. XTT colorimetric method test showed that the viability of P aeruginosa quorum sensing defective strain (P.a ΔlasI/rhlI)+ A fumigatus (A.f) was significantly higher than the wild-type P aeruginosa (PAO1)+ A fumigatus (A.f)(P<0.05). The electron microscope and scanning electron microscope observation of the biofilm ultrastructure suggested that the A fumigatus hypha density in the group of P aeruginosa quorum sensing defective strain (P.a ΔlasI/rhlI)+ A fumigatus (A.f) was significantly higher than that of group PAO1 + A.f . The hyphal density in the group of the wild-type P aeruginosa (PAO1)+ A fumigatus was less than the group with A. f alone . CONCLUSION The quorum sensing system of P aeruginosa inhibited the formation of early polymicrobial biofilms of the wild-type P aeruginosa (PAO1)+ A fumigatus.