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Open Access Highly Accessed Research article

Community analysis of bacteria colonizing intestinal tissue of neonates with necrotizing enterocolitis

Birgitte Smith1, Susan Bodé3, Bodil L Petersen4, Tim K Jensen2, Christian Pipper5, Julie Kloppenborg4, Mette Boyé2, Karen A Krogfelt1 and Lars Mølbak2*

Author Affiliations

1 Statens Serum Institut, Artillerivej 5, 2300 Kbh. S, Denmark

2 National Veterinary Institut- DTU, Bülowsvej 27, 1790 Copenhagen V, Denmark

3 Neonatel Department 5023, Rigshospitalet, Blegdamsvej, 2100 Kbh. Ø, Denmark

4 Pathologic Institut, Rigshospitalet, Blegdamsvej, 2100 Kbh. Ø, Denmark

5 Faculty of Life Sciences, University of Copenhagen, Bülowsvej 17, 1870 Frb., Denmark

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BMC Microbiology 2011, 11:73  doi:10.1186/1471-2180-11-73

Published: 12 April 2011

Abstract

Background

Necrotizing enterocolitis (NEC) is the most common gastrointestinal emergency in newborn neonates. Bacteria are believed to be important in the pathogenesis of NEC but bacterial characterization has only been done on human faecal samples and experimental animal studies. The aim of this study was to investigate the microbial composition and the relative number of bacteria in inflamed intestinal tissue surgically removed from neonates diagnosed with NEC (n = 24). The bacterial populations in the specimens were characterized by laser capture microdissection and subsequent sequencing combined with fluorescent in situ hybridization (FISH), using bacterial rRNA-targeting oligonucleotide probes.

Results

Bacteria were detected in 22 of the 24 specimens, 71% had moderate to high densities of bacteria. The phyla detected by 16S rRNA gene sequencing were: Proteobacteria (49.0%), Firmicutes (30.4%), Actinobacteria (17.1%) and Bacteroidetes (3.6%). A major detected class of the phylum Proteobacteria belonged to δ-proteobacteria. Surprisingly, Clostridium species were only detected in 4 of the specimens by FISH, but two of these specimens exhibited histological pneumatosis intestinalis and both specimens had a moderate to a high density of C. butyricum and C. parputrificum detected by using species specific FISH probes. A 16S rRNA gene sequence tag similar to Ralstonia species was detected in most of the neonatal tissues and members of this genus have been reported to be opportunistic pathogens but their role in NEC has still to be clarified.

Conclusion

In this study, in situ identification and community analysis of bacteria found in tissue specimens from neonates with NEC, were analysed for the first time. Although a large variability of bacteria was found in most of the analyzed specimens, no single or combination of known potential pathogenic bacteria species was dominating the samples suggestive NEC as non-infectious syndrome. However there was a significant correlation between the presence of C. butyricum & C. parputrificum and histological pneumatosis intestinalis. Finally this study emphasizes the possibility to examine the microbial composition directly on excised human tissues to avoid biases from faecal samples or culturing.

Keywords:
FISH; laser capture microdissection; microbiota; necrotizing enterocolitis; pneumatosis intestinalis; Ralstornia