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

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.

The role of the intestinal microbiota in the pathogenesis of necrotizing enterocolitis

“…When such diverse Contents lists available at SciVerse ScienceDirect journal homepage: www.elsevier.com/locate/sempedsurg Seminars in Pediatric Surgery methods have been used in the same study, they have yielded largely similar results. [3][4][5][6][7][8][9] Interestingly, little or no unculturable species of bacteria have been found, which supports the idea that culture methods miss relatively few, if any, bacterial species when it comes to analysis of the early microbiota. 10 Most of the studies examined fecal microbes.…”

“…These studies consistently found that regardless of what bacterial species appear first, there are relatively rapid changes in species diversity and overall load. 3,[5][6][7]9,11,16,18,21,[23][24][25] The first dominant colonizers are usually ''outcompeted'' by other species during the first 7-10 days of life. These new species in turn are overtaken by different groups of bacteria within a matter of weeks, and this succession pattern continues until the adult microbiota is established at about 1 year of age.…”

“…17 Other studies failed to establish any correlation between NEC and intestinal microbiota, prompting some authors to conclude that NEC is not associated with any infectious agent. 5,7,[33][34][35][36][37] Since the same bacterial species were found in both NEC patients and in healthy controls, no conclusion could be drawn as to the identity of the causative pathogens in infants who develop NEC. Although microbiome-wide studies might have revealed some patterns associated with NEC, they failed to unambiguously establish a relationship between the disease and the microbiota.…”

Gut microbiota of the very-low-birth-weight infant

“…Table 2 summarizes the molecular studies of GIT microbiota for VLBW infants with NEC or sepsis. In summary, these studies demonstrate lower bacterial diversity, less Bifidobacteria and Bacteroidetes, and a bloom of the γ-Proteobacteria 1-3 wk prior to the diagnosis of NEC Review (57)(58)(59)(60)(61)(62). A metagenomic analysis on stool samples from a set of twins discordant for the diagnosis of NEC demonstrated significant differences in the genes mapping to carbohydrate metabolism leading to speculation that microbial communities may metabolize milk through different pathways resulting in differing substrates available to the microbiota and thus differential effects on the host (57).…”

Molecular approaches for identification and characterization of lactic acid bacteria

“…54,55 DGGE and other similar techniques have been shown to be useful to analyze human fecal microflora. 56,57 For example, DGGE may be useful to assess the effect of antibiotic therapy on the fecal microflora of hospitalized patients. 58 Since the bacterial microbiota of different individuals may vary significantly the ability to provide a detailed analysis of multiple individuals is limited.…”