The Application Of Laser Capture Microdissection Combined With16s Rdna-dgge In Analysis Of Tissue Bacterial Floras

Microbial community diversity analysis for intestinal walltissue with neonatal necrotizing enterocolitis by comparing16S rDNA-DGGE with clinical culture【Objective】 To explore the microbial community diversity inintestinal wall tissue from patients neonatal necrotizing enterocolitis(NEC)by16S rDNA-denaturing gel gradient electrophoresis (DGGE),the resultswould be compared with clinical culture method.【Methods】The bacterial on pathological sections of18cases ofintestinal wall tissue from NEC patients and7cases of intestinal wall tissuefrom congenital intestinal atresia(CIA) patients were observed and recordedafter gram staining,then separated from the surrounding tissue by LaserCapture microdissection(LCM). After DNA extraction and PCRamplification, the microbial community diversity was analyzed with DGGE.【Results】The positive rate of bacterial culture was33.3%,4bacterial genera were detected in blood or other sterile fluids from18cases ofNEC,and each specimen contained range from0to2bacterial genera. Usingsequencing fragment of DNA,16bacterial genera mainly dominated byGram-negative bacterium were detected in18cases, each sample contained12genera on average,ranged from7to16, including cultivable anduncultivable bacteria.Protei composed mainly of γ-Protei,which accountedfor65.6%and Fimicutes accounted for30.5%of all the detected bacteria inintestinal wall tissue from NEC patients.Regarding to the controlgroup(intestinal wall tissue from CIA patients),10bacterial genera weredetected in7cases, each specimen ranged from1to6genera. Proteiaccounted for44.4%and Fimicutes accounted for50.0%of all the detectedbacteria. Pseudomonas spp,Klebsiella spp, Clostridium spp andAcinetobacter spp etc. could only be detected in the intestinal wall tissuefrom NEC patients.【Conclusion】Compared with the traditional culture method, LCMcombined with PCR-DGGE increase the detection rate of the pathogenicbacteria to investigate the microbial community diversity of inflammatorytissue. The microbiota in intestinal wall tissue from NEC patients is differentfrom the contron group.The proportion of Proteobacteria detected in NECgroup increased and the rate of Fimicutes decreased.Results that baterialonly detected in NEC group might contain the “potential pathogenicbacterium” could serve as a reference for effective application of antibiotics. Part IIComparative study on16S rDNA-DGGE versus conventionalclinical culture in analysis of children’s appendicitis tissuebacterial floras【Objective】To explore the microbial diversityinchildren’sappendixtissue with inflammation by PCR-denaturing gradient gelelectrophoresis(DGGE),compared with clinical culture method.【Methods】The bacterial on pathological sections of10cases ofappendicitis and4cases of normal appendix tissue were observed andrecorded after gram staining, thereafter separated from the surroundingtissue by Laser Capture Microdissection(LCM). After DNA extraction andPCR amplification, the microbial community diversity were analyzedthrough DGGE.【Results】The positive rate of bacterial culture is70%.4bacterialgenera were detected in10cases of appendicitis, and each specimencontained range from0to2bacterial genera. Using sequencing fragment ofDNA,14bacterial genera were detected in10cases, each sample contained9.6genera on average, ranged from6to12, including cultivable anduncultivable bacteria. Proteobacteria accounted for71.3%of all the detectedbacteria in appendicitis.Regarding to normal tissue, each specimen rangedfrom2to7genera, Proteobacteria accounted for60%of all the detectedbacteria. Pseudomonsa spp, Acinetobacter spp, Klebsiella spp and Serratia spp etc.could only be detected in the inflamatory tissue.【Conclusion】 LCM combined with PCR-DGGE increase thedetection rate of the pathogenic bacteria to analyze the microbial diversity ofinflammatory appendices tissue.It might help to provided more informationabout the actual condition of bacterial floras. The microbiota in inflamatoryappendix tissue is different from normal control tissue.Most of the florasdetected in appendicitis group were members of Proteobacteria which mightcontains the “potential pathogenic bacterium”.The results provide someevidence for reasonable choices of antibiotics.