Skip to main content
PMC home page
Journal of Clinical Microbiology logoLink to Journal of Clinical Microbiology
. 1996 Oct;34(10):2506–2510. doi: 10.1128/jcm.34.10.2506-2510.1996

Application of 16S rRNA gene PCR to study bowel flora of preterm infants with and without necrotizing enterocolitis.

M R Millar 1, C J Linton 1, A Cade 1, D Glancy 1, M Hall 1, H Jalal 1
PMCID: PMC229306  PMID: 8880510

Abstract

The purpose of the present study was to determine the extent to which bacteria not detected by culture contribute to the microbial flora of the bowel of preterm infants with and without neonatal necrotizing enterocolitis (NEC). Fecal samples from 32 preterm infants in special care baby units including samples from 10 infants with NEC were examined by culture and PCR amplification of the 16S rRNA gene (rDNA). The 16S rDNA V3 region was amplified with eubacterial primers, and the amplification products derived from the fecal sample DNA were compared with the products from individual cultured isolates by PCR and denaturing gradient gel electrophoresis (PCR-DGGE), allowing the DNA from uncultured bacteria to be identified. For the 22 infants without NEC weekly samples were examined for a mean of 5.3 postnatal weeks. The total number of types detected by culture combined with PCR-DGGE was 10.1 per infant, of which PCR-DGGE contributed 10.4% of the types identified. Additional types detected by PCR-DGGE were found in 14 (63.6%) of the 22 infants. The majority of the sequences associated with uncultured bacteria showed > 90% 16S rDNA sequence identity with sequences from culturable human enteric flora, and all were found in single infants with the exception of sequences indistinguishable by DGGE from seven infants. These sequences showed > 90% sequence identity with the 16S rDNA of Streptococcus salivarius and may have been derived from upper gastrointestinal or respiratory tract flora. In the present study uncultured bacteria detected by PCR-DGGE were no more frequent in fecal samples from infants with NEC than in samples from infants without NEC, although these findings do not exclude the possibility of unrecognized bacteria associated with the mucosa of the small intestine of infants with NEC.

Full Text

The Full Text of this article is available as a PDF (199.8 KB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Banas J. A., Simon D., Williams L. K., Ferretti J. J., Russell R. R. Analysis of a primer-independent GTF-I from Streptococcus salivarius. FEMS Microbiol Lett. 1994 Nov 1;123(3):349–354. doi: 10.1111/j.1574-6968.1994.tb07247.x. [DOI] [PubMed] [Google Scholar]
  2. Bennet R., Eriksson M., Nord C. E., Zetterström R. Fecal bacterial microflora of newborn infants during intensive care management and treatment with five antibiotic regimens. Pediatr Infect Dis. 1986 Sep-Oct;5(5):533–539. doi: 10.1097/00006454-198609000-00009. [DOI] [PubMed] [Google Scholar]
  3. Clark D. A., Thompson J. E., Weiner L. B., McMillan J. A., Schneider A. J., Rokahr J. E. Necrotizing enterocolitis: intraluminal biochemistry in human neonates and a rabbit model. Pediatr Res. 1985 Sep;19(9):919–921. doi: 10.1203/00006450-198509000-00010. [DOI] [PubMed] [Google Scholar]
  4. Crowther J. S. Transport and storage of faeces for bacteriological examination. J Appl Bacteriol. 1971 Jun;34(2):477–483. doi: 10.1111/j.1365-2672.1971.tb02307.x. [DOI] [PubMed] [Google Scholar]
  5. Dymock D., Weightman A. J., Scully C., Wade W. G. Molecular analysis of microflora associated with dentoalveolar abscesses. J Clin Microbiol. 1996 Mar;34(3):537–542. doi: 10.1128/jcm.34.3.537-542.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Eastick K., Leeming J. P., Bennett D., Millar M. R. Reservoirs of coagulase negative staphylococci in preterm infants. Arch Dis Child Fetal Neonatal Ed. 1996 Mar;74(2):F99–104. doi: 10.1136/fn.74.2.f99. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Fuhrman J. A., McCallum K., Davis A. A. Novel major archaebacterial group from marine plankton. Nature. 1992 Mar 12;356(6365):148–149. doi: 10.1038/356148a0. [DOI] [PubMed] [Google Scholar]
  8. Giovannoni S. J., Britschgi T. B., Moyer C. L., Field K. G. Genetic diversity in Sargasso Sea bacterioplankton. Nature. 1990 May 3;345(6270):60–63. doi: 10.1038/345060a0. [DOI] [PubMed] [Google Scholar]
  9. Glass R. I. New prospects for epidemiologic investigations. Science. 1986 Nov 21;234(4779):951–955. doi: 10.1126/science.3775371. [DOI] [PubMed] [Google Scholar]
  10. Goldmann D. A., Leclair J., Macone A. Bacterial colonization of neonates admitted to an intensive care environment. J Pediatr. 1978 Aug;93(2):288–293. doi: 10.1016/s0022-3476(78)80523-x. [DOI] [PubMed] [Google Scholar]
  11. Hoy C., Millar M. R., MacKay P., Godwin P. G., Langdale V., Levene M. I. Quantitative changes in faecal microflora preceding necrotising enterocolitis in premature neonates. Arch Dis Child. 1990 Oct;65(10 Spec No):1057–1059. doi: 10.1136/adc.65.10_spec_no.1057. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Itoh K., Freter R. Control of Escherichia coli populations by a combination of indigenous clostridia and lactobacilli in gnotobiotic mice and continuous-flow cultures. Infect Immun. 1989 Feb;57(2):559–565. doi: 10.1128/iai.57.2.559-565.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Kien C. L., Liechty E. A., Myerberg D. Z., Mullett M. D. Dietary carbohydrate assimilation in the premature infant: evidence for a nutritionally significant bacterial ecosystem in the colon. Am J Clin Nutr. 1987 Sep;46(3):456–460. doi: 10.1093/ajcn/46.3.456. [DOI] [PubMed] [Google Scholar]
  14. Kliegman R. M., Walker W. A., Yolken R. H. Necrotizing enterocolitis: research agenda for a disease of unknown etiology and pathogenesis. Pediatr Res. 1993 Dec;34(6):701–708. doi: 10.1203/00006450-199312000-00001. [DOI] [PubMed] [Google Scholar]
  15. Kosloske A. M. Epidemiology of necrotizing enterocolitis. Acta Paediatr Suppl. 1994;396:2–7. doi: 10.1111/j.1651-2227.1994.tb13232.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Maidak B. L., Larsen N., McCaughey M. J., Overbeek R., Olsen G. J., Fogel K., Blandy J., Woese C. R. The Ribosomal Database Project. Nucleic Acids Res. 1994 Sep;22(17):3485–3487. doi: 10.1093/nar/22.17.3485. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Millar M. R., Bacon C., Smith S. L., Walker V., Hall M. A. Enteral feeding of premature infants with Lactobacillus GG. Arch Dis Child. 1993 Nov;69(5 Spec No):483–487. doi: 10.1136/adc.69.5_spec_no.483. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Muyzer G., de Waal E. C., Uitterlinden A. G. Profiling of complex microbial populations by denaturing gradient gel electrophoresis analysis of polymerase chain reaction-amplified genes coding for 16S rRNA. Appl Environ Microbiol. 1993 Mar;59(3):695–700. doi: 10.1128/aem.59.3.695-700.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Myers R. M., Maniatis T., Lerman L. S. Detection and localization of single base changes by denaturing gradient gel electrophoresis. Methods Enzymol. 1987;155:501–527. doi: 10.1016/0076-6879(87)55033-9. [DOI] [PubMed] [Google Scholar]
  20. Pearson W. R. Rapid and sensitive sequence comparison with FASTP and FASTA. Methods Enzymol. 1990;183:63–98. doi: 10.1016/0076-6879(90)83007-v. [DOI] [PubMed] [Google Scholar]
  21. Pettersson B., Johansson K. E., Uhlén M. Sequence analysis of 16S rRNA from mycoplasmas by direct solid-phase DNA sequencing. Appl Environ Microbiol. 1994 Jul;60(7):2456–2461. doi: 10.1128/aem.60.7.2456-2461.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Relman D. A., Loutit J. S., Schmidt T. M., Falkow S., Tompkins L. S. The agent of bacillary angiomatosis. An approach to the identification of uncultured pathogens. N Engl J Med. 1990 Dec 6;323(23):1573–1580. doi: 10.1056/NEJM199012063232301. [DOI] [PubMed] [Google Scholar]
  23. Relman D. A., Schmidt T. M., MacDermott R. P., Falkow S. Identification of the uncultured bacillus of Whipple's disease. N Engl J Med. 1992 Jul 30;327(5):293–301. doi: 10.1056/NEJM199207303270501. [DOI] [PubMed] [Google Scholar]
  24. Sakata H., Yoshioka H., Fujita K. Development of the intestinal flora in very low birth weight infants compared to normal full-term newborns. Eur J Pediatr. 1985 Jul;144(2):186–190. doi: 10.1007/BF00451911. [DOI] [PubMed] [Google Scholar]
  25. Sneath P. H. Evidence from Aeromonas for genetic crossing-over in ribosomal sequences. Int J Syst Bacteriol. 1993 Jul;43(3):626–629. doi: 10.1099/00207713-43-3-626. [DOI] [PubMed] [Google Scholar]
  26. Wilson K. H., Blitchington R., Frothingham R., Wilson J. A. Phylogeny of the Whipple's-disease-associated bacterium. Lancet. 1991 Aug 24;338(8765):474–475. doi: 10.1016/0140-6736(91)90545-z. [DOI] [PubMed] [Google Scholar]
  27. Woese C. R. Bacterial evolution. Microbiol Rev. 1987 Jun;51(2):221–271. doi: 10.1128/mr.51.2.221-271.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Journal of Clinical Microbiology are provided here courtesy of American Society for Microbiology (ASM)

RESOURCES