Application Of 16S RDNA PCR - DGGE Combined With Sequencing In Neonatal Intestinal Microecology And Septicemia Pathogen Detection

BACTERIAL COMMUNITY COMPOSITION ASSOCIATED WITH CESAREAN DELIVERY VERSUS VAGINAL DELIVERY IN CHINESE NEWBORNSObjectives:To characterize the diversity of the intestinal microbiota in newborn infants delivered vaginally (VD) or with cesarean delivery (CD).Methods:A cross-sectional study was performed using fecal specimens collected on the 2nd day and 4th day of postnatal life from 25 VD infants and 16 CD infants. Profiles of the fecal microbiota were analyzed by PCR-denaturing gradient gel electrophoresis (DGGE) in combination with 16S rRNA gene sequencing of the clones corresponding to the DGGE bands.Results:On the 2nd day and 4th day of postnatal life, VD and CD infants did not differ statistically in the richness or evenness of the fecal bacterial community. The diversity indexs:Shannon indexs of VD and CD infants of day 2 were 1.94±0.29 and 2.30±0.25, the Simpson indexs were 0.15± 0.05 and 0.11±0.28; the Shannon evenness indexs were 0.95±0.25 and 0.97±0.15 respectively; the Shannon indexs of VD and CD infants of day 4 were 2.32±0.12 and 2.64±0.14, the Simpson indexs were 0.10±0.01 and 0.07±0.10; the Shannon evenness indexs were 0.98±0.08 and 0.99± 0.05. However, the taxa of the fecal microbiota were significantly different between the two groups (P>0.05). In VD infants, Escherichia coli (92.86%), Bacteroides sp. (78.57%), and Bifidobacterium longum(83.33%) were the dominant microbes. In CD infants, Staphylococcus sp. (72.73%), Clostridium sp. (45.45%), Enterobacter sp. (45.45%), and Streptococcus sp. (70.00%) were more common.Conclusions:These results demonstrate that delivery mode has a profound influence on the composition of the intestinal microbiota in Chinese newborn infants.IS THE 16S RDNA PCR-DGGE AND SEQUENCING METHOD EFFICIENT IN THE DIAGNOSIS OF NEONATAL LATE-ONSET SEPTICEMIA?Aim:To determine whether the 16S rDNA PCR-DGGE and sequencing method is efficient in detecting a comprehensive bacterial spectrum in neonatal late-onset septicemia.Methods:Blood specimens from 14 neonates whom septicemia was diagnosed were collected. Fourteen culture positive blood samples and 24 spiked "infected" blood samples were analyzed by the 16S rDNA PCR-DGGE and sequencing method or pathogen-specific PCR method in comparison with the blood culture method.Results:Only in 5 of the 14 cases did the results of 16S rDNA PCR-DGGE and sequencing method matched partly or wholly with blood culture. In the other 9 cases, blood culture failed to detect the bacteria such as Neisseria sp., Moraxella sp. which were detected by the 16S rDNA PCR-DGGE and sequencing method. Meanwhile, the 16S rDNA PCR-DGGE and sequencing technology also failed to detect the blood culture-proven bacteria, such as Klebsiella pneumonia, Enterococcus faecalis. Analysis of the reason leading to this discrepancy with pathogen-specific PCR and spiked "infected" blood samples indicated a competitive inhibitory effect in 16S rDNA PCR amplification. When detecting a certain species of bacteria (such as Enterococcus faecalis in the present study) by 16S rDNA PCR, the competitive inhibitory effect presented as a higher sensitivity in detecting Enterococcus faecalis DNA from the blood samples which contained Enterococcus faecalis DNA only (detection limit:8.32×10-4 4ng/μl) than the blood samples which were blended with Staphylococcus epidermidis and Pseudomonas aeruginosa DNA (detection limit:2.60 ng/μl).Conclusions:The 16S rDNA PCR-DGGE and sequencing method can detect a more comprehensive spectrum of pathogens than blood culture. However, the competitive inhibitory effect should be taken into consideration when the 16S rDNA PCR-DGGE and sequencing method is applied to the diagnosis of neonatal LOS which may lead to false negative results. PRIMARY INVESTIGATION OF EXOGENOUS BACTERIAL DNA CONTAMINATION IN DETECTION OFSEPTIC BACTERIAL SPECTRUM BY 16S RDNA PCR-DGGEAim:To investigate the exogenous bacterial DNA contamination in detection of septic bacterial spectrum by 16S rDNA PCR-DGGE.Methods:Blood specimens from patient diagnosed of septicemia and neonatal jaundice (not caused by infectious factors) were collected. The bacteria genomic DNA was then extracted with or without Lysozyme. The DGGE fingerprints of the blood specimens using the two different extraction methods were compared after 16S rDNA PCR-DGGE analysis.Results:DGGE fingerprints of the bacterial DNAs extracted from different blood samples using Lysozyme demonstrated identical bands. The sequencing results of these indetical bands were Vibrio sp., Escherichia coli and Aeromonas sp. The DGGE fingerprints of the DNAs extracted from the septic blood without the use of Lysozyme showed difference with the negative and bland controls which bacteria such as Staphylococcus epidermidis, Corynebacterium sp., Corynebacterium sp., Halomonas sp. and Propionibacterium sp. were detected. The amount and position of the bands on DGGE fingerprints from the negative and blank control were also different (14 bands and 17 bands respectively).Conclusions:Exogenous bacterial DNA in the Lysozyme solutions may be the primary reason for indentical bands both existed in experimental samples (septic blood) and the controls. This exogenous bacterial DNA contamination may be widely existed.