免疫印迹杂交在院内肺炎感染途径中的应用

目的：为确证胃→咽→下呼吸道逆行感染途径存在与否,研究胃液pH值变化与院内感染性肺炎(Nosocomial Pneumonia,NP)发生的关系。方法：以气管切开(或插管)病人为研究对象,用防污染标本刷(PSB)采集标本,由细菌表型分型,直至质粒DNA、酶切、Southern-blot印迹杂交等基因分型,前瞻性观察NP的发生情况。结果：11名患者有逆行感染存在(发生率为22％),即在胃部分离出病原菌1、2天后,PSB在咽部及下呼吸道也检出相同的细菌,应用质粒图谱、酶切图谱和Southern-blot印迹杂交等分子生物学技术进行同源性分析,证明其有很高的同源性。将美蓝经胃管注入病人胃内,1、2天后在咽部及下呼吸道可检出蓝色分泌物。随着胃液PH值升高,胃内细菌定植增加,NP的发生率升高。结论：存在胃→咽→下呼吸道逆行感染途径。NP的发生与胃液PH值显著相关。

Southern-blot analysis in the study of route of infection for nosocomial pneumonia

Objective To explore the evidence of retrograde route of transmission from stomach to oropharynx to lower respiratory tract and investigate the relationship between the incidence of nosocomial pneumonia and the pH value of the stomach.Methods The study was conducted requiring mechanical ventilation or tracheostomy,the secretion of lower respiratory tract was collected by protected specimen brush(PSB) and the specimens were carried out for quantitative culture of bacteria. At the same time, the bacterial samples of gastric aspirates were collected to culture and the pH value of gastric aspirates was determined using a digital acid base analyzer. All patients were monitored for development of nosocomial pneumonia every 2 days. The gene typings of the bacterial from lower respiratory tract secretion and gastric aspirates were take by plasmial profile analysis,restriction endonuclease fingerprint analysis and Southern-blot analysis in the same phenotype bacteria.Results In 11(22%) case of nosocomial pneumonia, the identical organisms were isolated first in the gastric samples and then one to two days later in tracheal secretion. They were very homologous,exhibiting the same fingerpriting in plasmid profile, endonuclease fingerprint and Southern blot analysis.Two days after being perfused into the stomach through masogastric tubes, Methylene blue was found in the secretion of throat and lower respiratory tract. The bacterial counts in gastric aspirates increased significantly with gastric pH.Conclusion The retrograde route of transmission is confirmed to be existed and it is one of the risk factors in nosocomial pneumonia.The occurrence of nosocomial pneumonia is significantly associated with rising of gastric pH.