机械通气患儿气管导管表面细菌生物膜形成及病原分析

The formation of bacterial biofilm and analysis of pathogens on tracheal tubes in ventilated children

目的观察儿科重症监护病房(PICU)机械通气(mechanical ventilation,MV)患儿气管导管(endotra-cheal tube,ETT)表面细菌生物膜(biofilm,BF)内细菌分布情况及BF形态学特征。方法以我院治疗的35例MV患儿为研究对象。收集第1次拔除或更换的ETT,经碘化丙啶(PI)和异硫氰酸荧光素标记刀豆蛋白A(FITC-ConA)染色后,激光共聚焦显微镜观察ETT-BF内细菌及胞外多糖(EPS)分布情况,并收集ETT表面和下呼吸道分泌物进行细菌分离、培养和鉴定。结果(1)35例MV患儿中,ETT表面细菌培养阳性31例(88.57%);17例ETT表面和下呼吸道分泌物同时分离出相同菌种,占ETT培养阳性的54.83%。在ETT-BF和下呼吸道分泌物中以金黄色葡萄球菌,肺炎克雷伯菌,大肠埃希菌,阴沟肠杆菌最常见。(2)CLSM观察可见,气管插管12 h后,ETT表面已出现细菌黏附;48 h时ETT表面可见大量短棒状或球状细菌黏附聚集,EPS较12 h时显著增多,初步形成BF结构;72 h左右细菌粘连成团块状,被EPS包裹,可见成熟BF结构形成;7 d后细菌粘连成大片状,在其周围可见散在的微菌落。(3)35例MV患儿中,19例发生了呼吸机相关性肺炎(ventilated-associated pneumonia,VAP)。其中经口插管10例,经鼻插管9例。结论MV时细菌极易在ETT表面黏附,形成细菌BF。ETT表面细菌定植及BF形成与长时间MV患儿伴发呼吸机相关性肺炎之间可能存在一定相关性。

Objective To observe the distribution of bacteria and morphologic features of biofilms （BFs） on the surface of endotracheal tubes （ETT） in PICU. Method 35 children undergoing mechanical ventilation （MV） were in- volved in this study. ETrs were collected at the first time of extubation. After staining with PI and FITC-ConA, the distribu- tion of the bacteria and extracellular polymeric substances （EPS） were observed by cenfocal laser scanning microscopy （CLSM）. Meanwhile,bacteria in BF and the secretions of lower respiratory tract were isolated, cultured and identified,re- spectively. Result （ 1 ） Of 35 EITs, 31 tubes were positive （88.57%）. And 17 samples had the same pathogen both in the ETTs and secretions of lower respiratory tract, which accounted for 54. 83% of the positive cultures from ETTs. Staphylococcus aureu,Klebsiella pneumoniae, Escherichia coli and Enterobacter cloacae were the main pathogens in BF and the secretions of lower respiratory tract. （2） Observation by CLSM showed that the bacteria were adhered on the surface of the ETrs in 12 hours after intubation; after 48 hours, lots of spherical or clubbed bacteria adhered into clusters, EPS al- so increased, and the initial BF was formed; After 72 hours, bacteria which encased in mass of EPS aggregated mutually into clumps, and formed mature BF; After more than 7 days the bacteria adhered into lamellars, and there were some mi- crocolonies around mature BF. （3） Of 35 mechanical ventilated children, 19 cases had VAP, including 10 in orotracheal in- tubation and 9 in nasotracheal intubation. Conclusion Bacterial biofilm can form on the surface of ETTs when ventilated. There may be correlation between microbial colonization and BF formation on the surface of ETrs and VAP in children who were intubated and ventilated for a long period.