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

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.

Figure 1

Epifluorescence micrographs of fluorescent in situ hybridized tissue samples taken from neonates diagnosed with necrotizing enterocolitis. All the specimens were hybridized with a general bacterial probe (EUB338) tagged with fluorescein (green colour) and group specific probes tagged with Cy3 (red colour). A) Visualization of Enterobacteria (GAM42a). B) Visualization of Actinobacteria ( pB00182). C) Visualization of Clostridium butyricum ( S-S-C. butyricum-663) in the two neonates where pneumatosis intestinalis was verified by histopathology. D) Visualization of Clostridium perfringens (S-S-C.perfring-185-a-A-18) in neonate number 3 with pneumatosis intestinalis.The scale bar is 20 μm in all the micrographs.

Figure 2

The total bacterial composition from eight intestinal tissue samples by 16S rRNA gene clone library. The γ-Proteobacteria dominated the total bacterial composition whereas the class Clostridia only accounted for a total of 7.1%

Figure 3

Overview and diversity of the bacterial composition by clone library analysis. a) Shannon's diversity index on phylum level divided the NEC infants in two groups. This difference could not be explained by antibiotic treatments or the severity of the necrotizing enterocolitis b) The bacterial 16S rRNA gene composition from each of the eight necrotic intestinal tissue samples. Bacterial groups whose abundance were more than 10% in any sample are shown as bars. Enterococcus and Escherichia spp. were the most abundant in the samples with a low Shannon diversity index where Ralstonia sp. was the most frequent group of species in the samples with a high Shannon index.

Figure 4

Phylogenetic relationship among Ralstonia detected in the tissue samples from the NEC infants. R. detusculanense, R. pickettii and R. insidiosa did all have more than 99% similarity with the matched Ralstonia tag from the 16S rRNA gene clone library from this study. The bacteria names and the accession numbers are shown.