CAJ | 학술논문

目的观察机械通气新生儿气管导管表面生物膜细菌群落多样性,探讨以变性梯度凝胶电泳(DGGE)技术为基础的细菌鉴定方法与传统培养法的差异,为临床预防、诊疗细菌性感染提供新思路.方法以重庆医科大学附属儿童医院新生儿中心2011年10月至2012年3月经治的26例患儿35根气管导管为研究对象,依靠16S rDNA PCR-DGGE技术,通过quantity one软件分析气管导管表面生物膜中细菌群落多样性,通过T-A克隆测序,了解生物膜中细菌种类分布及常见优势菌.结果 35根气管导管经16S rDNA PCR-DGGE所得条带13.8±5.4,香农威纳指数2.42 ±0.38.16份标本来自7例患儿不同疾病阶段,其中6例患儿气管导管留置天数增加,香农威纳指数减小.6例不同基础疾病患儿的气管导管标本香农威纳指数各不相同.回收24条优势条带克隆测序.35份标本中检出克雷伯菌属100％、假单胞菌属80％、链球菌属77％,91％的标本中存在无法培养的细菌,97％检测到2种以上菌属.80％克雷伯菌属和82％链球菌属中分别与假单胞菌属伴随存在.22例有临床痰培养结果,肺炎克雷伯菌10例(45％),鲍曼不动杆菌2例(9％),阴沟肠杆菌、阴性结果各1例(5％),培养结果均为单菌属.8例痰培养显示“正常菌群”,其对应导管上均检测到克雷伯菌等菌属.结论气管导管表面生物膜中细菌群落具有多样性；16S rDNA PCR-DGGE技术相比培养法更能全面反映气管导管表面细菌群落组成和多样性；克雷伯菌属、假单胞菌属及链球菌属是气管导管表面生物膜中常见菌属,它们在生物膜形成过程中可能存在相互作用.
목적 관찰궤계통기신생인기관도관표면생물막세균군락다양성,탐토이변성제도응효전영(DGGE)기술위기출적세균감정방법여전통배양법적차이,위림상예방、진료세균성감염제공신사로.방법 이중경의과대학부속인동의원신생인중심2011년10월지2012년3월경치적26례환인35근기관도관위연구대상,의고16S rDNA PCR-DGGE기술,통과quantity one연건분석기관도관표면생물막중세균군락다양성,통과T-A극륭측서,료해생물막중세균충류분포급상견우세균.결과 35근기관도관경16S rDNA PCR-DGGE소득조대13.8±5.4,향농위납지수2.42 ±0.38.16빈표본래자7례환인불동질병계단,기중6례환인기관도관류치천수증가,향농위납지수감소.6례불동기출질병환인적기관도관표본향농위납지수각불상동.회수24조우세조대극륭측서.35빈표본중검출극뢰백균속100％、가단포균속80％、련구균속77％,91％적표본중존재무법배양적세균,97％검측도2충이상균속.80％극뢰백균속화82％련구균속중분별여가단포균속반수존재.22례유림상담배양결과,폐염극뢰백균10례(45％),포만불동간균2례(9％),음구장간균、음성결과각1례(5％),배양결과균위단균속.8례담배양현시“정상균군”,기대응도관상균검측도극뢰백균등균속.결론 기관도관표면생물막중세균군락구유다양성；16S rDNA PCR-DGGE기술상비배양법경능전면반영기관도관표면세균군락조성화다양성；극뢰백균속、가단포균속급련구균속시기관도관표면생물막중상견균속,타문재생물막형성과정중가능존재상호작용.
Objective The catheter-related infections caused by mechanical ventilation have become a intractable clinical problem,and it is related to the formation of bacterial biofilm (BF) on the surface of the implanted material.The majority of natural biofilms are formed by multiple bacterial species.However,there always only one or limited species were detected on tracheal tubes removed from intubated neonates by using traditional methods including bacterium culture and antigen detection.The aims of this study were to observe the bacterial communities diversity of BF on endotracheal tube (ETT),and discuss the difference between traditional bacterium culture methods and the use of molecular biology techniques on the basis of denatured gradient gel electrophoresis (DGGE),to provide new ideas for clinical prevention,diagnosis and treatment of bacterial infections.Method Thirty-five ETTs were obtained from 26 neonates on mechanical ventilator (from October 2012 to March 2013) in Department of Neonatology of Children's Hospital.Among the patients,18 were boys and 8 girls,and 19 patients were ＜37 weeks gestational age and 7 patients ≥37 weeks.DGGE profiling of 16S rDNA gene amplicons was used to assess the diversity of the bacterial population by using the software of quantity one.TA Cloning Kit and sequencing were used to investigate the distribution of bacteria and common dominant bacteria in ETT-BF.Result The mean bands of 35 ETTs cases were 13.8 ± 5.4 from 16S rDNA PCR-DGGE,and the mean Shanon-wiener indexes was 2.42 + 0.38.The 16 ETTs were collected in different stages of diseases from the 7 patients.The indwelling days of 6/7 patients' ETTs increased,the Shanon-wiener indexes were decreased.Among the 6 cases from different basic illnesses,and there were different Shanon-wiener indexes.The result of molecular cloning and sequencing for 24 dominant bands showed that 35 cases (100％) contained Klebsiella SP.,28 cases (80％) had Pseudomonas SP.,27 cases (77％) had Streptococcus SP.,and 32 cases (91％) had Uncultured bacterium,while more than 2 bacterial species were found in 34 cases (97％).28/35 (80％)Klebsiella SP.and 22/27 (82％) Streptococcus SP.were accompanied by Pseudomonas SP.There were 22 positive results of sputum culture from 26 newborns,including 10 strains (45％) of Klebsiella pneumoniae,2 strains (9％) of Acinetobacter baumannii,Enterobacter cloacae and non-cultured bacterium in each patient (5％),but only one bacterium isolated from every sputum.Eight sputum samples had normal flora only,corresponding to the ETTs on which Klebsiella and other bacterial genuses were found.Conclusion The diversity of microbiota in BF on ETT was confirmed.16S rDNA PCR-DGGE could produce a more complete picture of bacterial community than traditional bacterium culture method.Klebsiella,Pseudomonas and Streptococcus were common dominant bacteria in ETT-BF,and there might be interactions among them in the formation of BF.