16S rRNA gene-based analysis of fecal microbiota from preterm infants with and without necrotizing enterocolitis

Yunwei Wang, Jeanette D Hoenig, Kathryn J Malin, Sanaa Qamar, Elaine O Petrof, Jun Sun, Dionysios A Antonopoulos, Eugene B Chang, Erika C Claud, Yunwei Wang, Jeanette D Hoenig, Kathryn J Malin, Sanaa Qamar, Elaine O Petrof, Jun Sun, Dionysios A Antonopoulos, Eugene B Chang, Erika C Claud

Abstract

Neonatal necrotizing enterocolitis (NEC) is an inflammatory intestinal disorder affecting preterm infants. Intestinal bacteria have an important function; however no causative pathogen has been identified. The purpose of this study was to determine if there are differences in microbial patterns that may be critical to the development of this disease. Fecal samples from 20 preterm infants, 10 with NEC and 10 matched controls (including 4 twin pairs) were obtained from patients in a single site level III neonatal intensive care unit. Bacterial DNA from individual fecal samples was PCR-amplified and subjected to terminal restriction fragment length polymorphism analysis and library sequencing of the 16S rRNA gene to characterize diversity and structure of the enteric microbiota. The distribution of samples from NEC patients distinctly clustered separately from controls. Intestinal bacterial colonization in all preterm infants was notable for low diversity. Patients with NEC had even less diversity, an increase in abundance of Gammaproteobacteria, a decrease in other bacteria species, and had received a higher mean number of previous days of antibiotics. Our results suggest that NEC is associated with severe lack of microbiota diversity that may accentuate the impact of single dominant microorganisms favored by empiric and widespread use of antibiotics.

Figures

Figure 1. Diversity comparison between preterm infants…
Figure 1. Diversity comparison between preterm infants with and without NEC by terminal restriction fragment length polymorphism (T-RFLP) and clone library analysis
(a and b) Representative T-RFLP profiles of intestinal bacteria in infants with and without NEC. N1 (with NEC) and C1 (without NEC) were samples collected from a genetically identical twin pair. CN10 and N10 were samples collected from the same infant before and after the development of NEC. For each profile, the number of peaks (N) and Shannon diversity index (S) are shown. (c) Overall Shannon diversity index between NEC and control groups was calculated from T-RFLP data based on peak number and distribution in T-RFLP profiles from each sample and shows decreased diversity in the NEC patients. (d) Overall diversity by library cloning and sequencing analysis in preterm infants with and without NEC again demonstrates decreased diversity in NEC patients. (e) Comparison of antibiotic exposure time between control and NEC groups. In all panels, * indicates statistical significance at P < 0.05. NEC, necrotizing enterocolitis.
Figure 2. Overview of bacterial composition of…
Figure 2. Overview of bacterial composition of all samples by clone library analysis
(a) Relative abundance of Proteobacteria in preterm infants. * Comparison of Proteobacteria between two groups, P < 0.01; # Comparison of other phyla (Firmicutes for NEC; Firmicutes, Bacteroidetes and Fusobacteria for control) between the two groups, P < 0.01. (b and c) The genus-level composition of gut microbiota. Genera whose abundance were more than 10% in any sample and its corresponding phyla (P: Proteobacteria; F: Firmicutes; B: Bacteroidetes) are shown as bars (NEC samples top panel, Control samples bottom panel). The dominant genus in each sample is labeled. Only samples from NEC infants had dominance of a single genus of the Proteobacteria phylum (more than 50%). Samples C9 and CN10 are notable for Escherichia dominance, and developed NEC shortly after samples were obtained. NEC, necrotizing enterocolitis.
Figure 3. Phylogenetic relationships among the OTUs…
Figure 3. Phylogenetic relationships among the OTUs detected in fecal samples both from control and NEC infants
The representative 125 clone numbers are listed in the tree. Clones labeled with ECC on blue branches were obtained from control infants. Clones labeled with ECN on red branches were obtained from NEC infants. Sequences with a more than 99% match are given the name of the matched species in GenBank and labeled with the number of identified clones in the libraries. The accession number, the nearest neighbor, the level of similarity and the number of clones in the clone library for each sequence are shown in Supplementary Table 1 and Table 2. Bootstrap values are based on 1000 replications and values above 50% are labeled. OTUs, operational taxonomical units.
Figure 4
Figure 4
Decreased diversity (a) and increased abundance of Proteobacteria (b) are demonstrated between samples collected from infants with and without NEC in samples from 4 sets of twins. (c) Dendrogram demonstrating that even for twin pairs, NEC patients (N1–N4, red) clustered together and distinctly separated from their healthy cohorts (C1–C4, blue) by UniFrac analysis. NEC, necrotizing enterocolitis.
Figure 5. Principal coordinate analysis (PCA) of…
Figure 5. Principal coordinate analysis (PCA) of sequence libraries
Samples collected from infants without NEC (control) are represented by squares (blue); samples from infants with NEC are represented by circles (red). Distribution of samples collected from infants with NEC was distinct from that collected from control infants. Samples C9 and CN10 which were collected from control patients who later developed NEC, notably clustered with the NEC group. NEC, necrotizing enterocolitis.

References

    1. Bell MJ, Feigin RD, Ternberg JL. Changes in the incidence of necrotizing enterocolitis associated with variation of the gastrointestinal microflora in neonates. Am J Surg. 1979;138(5):629–631.
    1. Bell MJ, Shackelford P, Feigin RD, Ternberg JL, Brotherton T. Epidemiologic and bacteriologic evaluation of neonatal necrotizing enterocolitis. J Pediatr Surg. 1979;14(1):1–4.
    1. Bell MJ, Ternberg JL, Feigin RD, Keating JP, Marshall R, Barton L, et al. Neonatal necrotizing enterocolitis. Therapeutic decisions based upon clinical staging. Ann Surg. 1978;187(1):1–7.
    1. Bin-Nun A, Bromiker R, Wilschanski M, Kaplan M, Rudensky B, Caplan M, et al. Oral probiotics prevent necrotizing enterocolitis in very low birth weight neonates. J Pediatr. 2005;147(2):192–196.
    1. Chang JY, Antonopoulos DA, Kalra A, Tonelli A, Khalife WT, Schmidt TM. Decreased diversity of the fecal Microbiome in recurrent Clostridium difficile-associated diarrhea. J Infect Dis. 2008;197(3):435–438.
    1. Claud EC, Walker WA. Hypothesis: inappropriate colonization of the premature intestine can cause neonatal necrotizing enterocolitis. Faseb J. 2001;15(8):1398–1403.
    1. Conte MP, Schippa S, Zamboni I, Penta M, Chiarini F, Seganti L, et al. Gut-associated bacterial microbiota in paediatric patients with inflammatory bowel disease. Gut. 2006;55(12):1760–1767.
    1. Cotten CM, Taylor S, Stoll B, Goldberg RN, Hansen NI, Sanchez PJ, et al. Prolonged duration of initial empirical antibiotic treatment is associated with increased rates of necrotizing enterocolitis and death for extremely low birth weight infants. Pediatrics. 2009;123(1):58–66.
    1. Dicksved J, Halvarson J, Rosenquist M, Jarnerot G, Tysk C, Apajalahti J, et al. Molecular analysis of the gut microbiota of identical twins with Crohn’s disease. ISME. 2008;2(7):716–727.
    1. Eckburg PB, Bik EM, Bernstein CN, Purdom E, Dethlefsen L, Sargent M, et al. Diversity of the human intestinal microbial flora. Science. 2005;308(5728):1635–1638.
    1. Gewolb IH, Schwalbe RS, Taciak VL, Harrison TS, Panigrahi P. Stool microflora in extremely low birthweight infants. Arch Dis Child Fetal Neonatal Ed. 1999;80(3):F167–F173.
    1. Goldmann DA, Leclair J, Macone A. Bacterial colonization of neonates admitted to an intensive care environment. J Pediatr. 1978;93(2):288–293.
    1. Good IJ. The population frequencies of species and the estimation of population parameters. Biometrika. 1953:237–264.
    1. Gronlund MM, Lehtonen OP, Eerola E, Kero P. Fecal microflora in healthy infants born by different methods of delivery: permanent changes in intestinal flora after cesarean delivery. J Pediatr Gastroenterol Nutr. 1999;28(1):19–25.
    1. Harmsen HJ, Wildeboer-Veloo AC, Raangs GC, Wagendorp AA, Klijn N, Bindels JG. Analysis of intestinal flora development in breast-fed and formula-fed infants by using molecular identification and detection methods. J Pediatr Gastroenterol Nutr. 2000;30(1):61–67.
    1. Hooper LV, Bry L, Falk PG, Gordon JI. Host-microbial symbiosis in the mammalian intestine: exploring an internal ecosystem. Bioessays. 1998;20(4):336–343.
    1. Hooper LV, Wong MH, Thelin A, Hansson L, Falk PG, Gordon JI. Molecular analysis of commensal host-microbial relationships in the intestine. Science. 2001;291(5505):881–884.
    1. Hoyos AB. Reduced incidence of necrotizing enterocolitis associated with enteral administration of Lactobacillus acidophilus and Bifidobacterium infantis to neonates in an intensive care unit. Int J Infect Dis. 1999;3(4):197–202.
    1. Huber T, Faulkner G, Hugenholtz P. Bellerophon: a program to detect chimeric sequences in multiple sequence alignments. Bioinformatics. 2004;20(14):2317–2319.
    1. Kosloske AM. Epidemiology of necrotizing enterocolitis. Acta Paediatr Suppl. 1994;396:2–7.
    1. Kuehl CJ, Wood HD, Marsh TL, Schmidt TM, Young VB. Colonization of the cecal mucosa by Helicobacter hepaticus impacts the diversity of the indigenous microbiota. Infect Immun. 2005;73(10):6952–6961.
    1. Kumar S, Nei M, Dudley J, Tamura K. MEGA: a biologist-centric software for evolutionary analysis of DNA and protein sequences. Brief Bioinform. 2008;9(4):299–306.
    1. Lepage P, Seksik P, Sutren M, de la Cochetiere MF, Jian R, Marteau P. Biodiversity of the mucosa-associated microbiota is stable along the distal digestive tract in healthy individuals and patients with IBD. Inflamm Bowel Dis. 2005;11(5):473–480.
    1. Lin HC, Su BH, Chen AC, Lin TW, Tsai CH, Yeh TF. Oral probiotics reduce the incidence and severity of necrotizing enterocolitis in very low birth weight infants. Pediatrics. 2005;115(1):1–4.
    1. Lozupone C, Hamady M, Knight R. UniFrac—an online tool for comparing microbial community diversity in a phylogenetic context. BMC Bioinformatics. 2006;7:371.
    1. Lozupone C, Knight R. UniFrac: a new phylogenetic method for comparing microbial communities. Appl Environ Microbiol. 2005;71(12):8228–8235.
    1. Lucas A, Cole TJ. Breast milk and neonatal necrotising enterocolitis. Lancet. 1990;336(8730):1519–1523.
    1. Millar MR, MacKay P, Levene M, Langdale V, Martin C. Enterobacteriaceae and neonatal necrotising enterocolitis. Arch Dis Child. 1992;67(1 Spec No):53–56.
    1. Palmer C, Bik EM, Digiulio DB, Relman DA, Brown PO. Development of the Human Infant Intestinal Microbiota. PLoS Biol. 2007;5(7):e177.
    1. Panigrahi P, Gupta S, Gewolb IH, Morris JG., Jr Occurrence of necrotizing enterocolitis may be dependent on patterns of bacterial adherence and intestinal colonization: studies in Caco-2 tissue culture and weanling rabbit models. Pediatr Res. 1994;36(1 Pt 1):115–121.
    1. Penders J, Thijs C, Vink C, Stelma FF, Snijders B, Kummeling I, et al. Factors influencing the composition of the intestinal microbiota in early infancy. Pediatrics. 2006;118(2):511–521.
    1. Relman DA. The search for unrecognized pathogens. Science. 1999;284(5418):1308–1310.
    1. Salyers AA, West SE, Vercellotti JR, Wilkins TD. Fermentation of mucins and plant polysaccharides by anaerobic bacteria from the human colon. Appl Environ Microbiol. 1977;34(5):529–533.
    1. Schloss PD, Handelsman J. Introducing DOTUR, a computer program for defining operational taxonomic units and estimating species richness. Appl Environ Microbiol. 2005;71(3):1501–1506.
    1. Schwiertz A, Gruhl B, Lobnitz M, Michel P, Radke M, Blaut M. Development of the intestinal bacterial composition in hospitalized preterm infants in comparison with breast-fed, full-term infants. Pediatr Res. 2003;54(3):393–399.
    1. Stappenbeck TS, Hooper LV, Gordon JI. Developmental regulation of intestinal angiogenesis by indigenous microbes via Paneth cells. Proc Natl Acad Sci U S A. 2002;99(24):15451–15455.
    1. Stoll BJ. Epidemiology of necrotizing enterocolitis. Clin Perinatol. 1994;21(2):205–218.
    1. Sturm R, Staneck JL, Stauffer LR, Neblett WW., 3rd Neonatal necrotizing enterocolitis associated with penicillin-resistant, toxigenic Clostridium butyricum. Pediatrics. 1980;66(6):928–931.
    1. Thompson JD, Gibson TJ, Plewniak F, Jeanmougin F, Higgins DG. The CLUSTAL_X windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Res. 1997;25(24):4876–4882.
    1. Turnbaugh PJ, Ley RE, Mahowald MA, Magrini V, Mardis ER, Gordon JI. An obesity-associated gut microbiome with increased capacity for energy harvest. Nature. 2006;444(7122):1027–1031.
    1. Vercellotti JR, Salyers AA, Bullard WS, Wilkins D. Breakdown of mucin and plant polysaccharides in the human colon. Can J Biochem. 1977;55(11):1190–1196.
    1. Young VB, Schmidt TM. Antibiotic-associated diarrhea accompanied by large-scale alterations in the composition of the fecal microbiota. J Clin Microbiol. 2004;42(3):1203–1206.

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