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Usefulness of denaturing high-pressure liquid chromatography to assess in situ gut microbiota in neonates

Abstract number: P918

De La Cochetiere M.F., Caillaux G., Le Vacon F., Caillon J., Rozé J.C., Potel G., Gras-Le Guen C.

Objectives: Microbiota colonisation of the neonatal gastrointestinal tract develops within a few hours after birth and major changes occur during the first postnatal weeks. Current knowledge of the gut microbial ecology is based on faeces studies, mainly by classical culture-dependent techniques. Nowadays, a new technique of fingerprinting, the DHPLC can be used. So far, however, no data is available for kinetics of implantation, growth and development of endoluminal microbiota in infants. Thus we did a prospective analysis of the in situ gut microbiota during neonatal surgery by classical cultures techniques, TTGE and DHPLC.

Methods: Newborns under 28 days of life requiring digestive surgery were included after parental information. During surgery, intestinal swabs were sampled. Then, the intestinal microbiota was analysed by conventional culture-based techniques and by two culture independent methods based upon bacteria DNA extraction and separation of amplified 16S rRNA gene fragments using Denaturing High Pressure Liquid Chromatography (DHPLC) and Temporale Temperature-Gradient Gel Electrophoresis (TTGE).

Results: 11 newborns (gestational age 33±2 weeks) were included. Two were born via caesarean section. Among the 6 newborns operated before 48 hours of life, 4 had a negative culture, no TTGE bands, three of which had a positive DHPLC analysis. The most frequent bacteria were Gram-negative bacilli (Enterobacter and E. coli), Gram-positive cocci (negative coagulase Staphylococcus-NCoS). Three were found with Pseudomonas (P. aeruginosa) and one with Clostridium (C. butyricum).

The three techniques used have shown coherent results. None uncultivable bacteria have been found.

Conclusion: Our work shows that DHPLC, based on DNA static analysis, is a powerful technology. DHPLC is a reproductible method that allows rapid characterisation of the bacterial establishment in neonates. DNA amplicons are easy to elute for sequencing. Moreover, it offers full automation. The neonatal supra-mesocolic tractus is colonised very early in life mostly with Enterobacteria and NCoS.

These results could be helpful to design specific antibioprophylaxis guidelines in neonates and to monitor microbiota changes induced by various neonatal diets and treatments.

This work has been supported by Atlangene and Transgenomic

Session Details

Date: 19/04/2008
Time: 00:00-00:00
Session name: 18th European Congress of Clinical Microbiology and Infectious Diseases
Subject:
Location: Barcelona, Spain
Presentation type:
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