Acute necrotizing enterocolitis of preterm piglets is characterized by dysbiosis of ileal mucosa-associated bacteria - PubMed (original) (raw)

Acute necrotizing enterocolitis of preterm piglets is characterized by dysbiosis of ileal mucosa-associated bacteria

M Andrea Azcarate-Peril et al. Gut Microbes. 2011 Jul-Aug.

Abstract

Investigation of bacteria involved in pathogenesis of necrotizing enterocolitis (NEC) is limited by infant fragility, analysis restricted to feces, use of culture-based methods, and lack of clinically-relevant animal models. This study used a unique preterm piglet model to characterize spontaneous differences in microbiome composition of NEC-predisposed regions of gut. Preterm piglets (n=23) were cesarean-delivered and nurtured for 30 hours over which time 52% developed NEC. Bacterial DNA from ileal content, ileal mucosa, and colonic mucosa were PCR amplified, subjected to terminal restriction fragment length polymorphism (TRFLP) analysis and targeted 16S rDNA qPCR. Preterm ileal mucosa was specifically bereft in diversity of bacteria compared to ileal content and colonic mucosa. Preterm ileum was restricted to representation by only Proteobacteria, Firmicutes, Cyanobacteria and Chloroflexi. In piglets with NEC, ileal mucosa was uniquely characterized by increases in number of Firmicutes and diversity of phyla to include Actinobacteria and uncultured bacteria. Five specific TRFLP profiles, corresponding in closest identity to Clostridium butyricum, C. neonatale, C. proteolyticum, Streptomyces spp., and Leptolyngbya spp., were significantly more prevalent or observed only among samples from piglets with NEC. Total numbers of Clostridium spp. and C. butyricum were significantly greater in samples of NEC ileal mucosa but not ileal content or colonic mucosa. These results provide strong support for ileal mucosa as a focus for investigation of specific dysbiosis associated with NEC and suggest a significant role for Clostridium spp., and members of the Actinobacteria and Cyanobacteria in the pathogenesis of NEC in preterm piglets.

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Figures

Figure 1

Figure 1

Criteria for diagnosis of NEC in preterm, cesarean-derived and formula-fed piglets. Criteria for NEC included severe abdominal distension, gaseous distention of the intestines (A; closed arrow = gas in portal vein; open arrow = orogastric feeding tube), hemorrhagic discoloration and sloughing or ulceration of the small or large bowel and atrophy, necrosis and sloughing of epithelium (B).

Figure 2

Figure 2

Phylum-associated unique TRFLP profiles expressed as percent of total unique TRFLP profiles identified by disease condition (NEC or No NEC) and sampling site (ileal mucosa, ileal lumen content and colonic mucosa) of piglets. The analysis shows a very highly diverse population of Proteobacteria, represented by the number of unique patterns associated with this phylum, compared to the rest of detected phyla. n = number of piglets.

Figure 3

Figure 3

Principal Coordinate Analysis plots of unique TRFLP patterns as grouped by disease condition (A; 22 no NEC [white] and 27 NEC [black] samples), sex of piglet (B; 36 female [white] and 11 male [black] samples) and sample location (C; 24 ileum [white] and 25 colon [black] mucosa).

Figure 4

Figure 4

Quantitative PCR for total Eubacterial 16s rDNA (A; *p < 0.05 versus ileal mucosa) and Clostridium genus and species-specific 16s rDNA (B; *p < 0.05 versus no NEC). Number of samples = ileal mucosa (no NEC, 11; NEC, 13), ileal content (no NEC, 9; NEC, 10) and colonic mucosa (no NEC, 11; NEC, 14). Data represent median, 10th, 25th, 75th and 90th percentiles.

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