The healthy adult human harbors roughly 1×1014
bacteria in the intestinal tract and these organisms make up about 60% of the total fecal mass.
Derangements in the numbers or types of bacteria can affect health and influence disease states. While human data are just starting to emerge, animal studies have suggested that microbiota composition in the neonatal period may affect gastrointestinal (GI) tract development, mucosal integrity, and even nutritional status.
Thus, developing an understanding of the neonatal (especially the very preterm) microbiota would be invaluable in helping to elucidate the processes that promote health or alternatively predispose to diseases, such as NEC.
In contrast to an earlier report that suggested reduced microbiota diversity in samples collected from cases after NEC diagnosis and a distinct clustering when compared with matched controls,
we did not detect such differences in overall diversity measures in our study. Rather, our findings confirm and extend our earlier observation that overall microbiota diversity at the time of diagnosis does not differ between healthy preterm infants and infants with NEC.
Our observation that one week before diagnosis microbial structure was more heterogeneous in infants that developed NEC suggests the potential for early detection of risk and focused intervention. Based on the data presented here it is feasible that future studies that include more cases will allow for the identification of a “high risk” microbiota pattern based on discriminant analysis of 16S rRNA sequences.
We used high throughput sequencing in a prospective study design. Our sequence depth per subject was more than 20 times deeper than that previously reported 
allowing us to detect rare OTUs that would have been missed with less extensive sequencing. We did confirm the presence of a higher proportion of Proteobacteria
that the same group reported only after the diagnosis of NEC. 
Furthermore, we observed that the proportion of Proteobacteria
increased in samples collected from NEC infants over the week prior to the diagnosis of NEC. In the samples collected from the NEC infants one week before diagnosis, the proportion of Proteobacteria
was actually lower than that observed in the matched control samples. Hence, our data raise the possibility that preterm infants not sufficiently colonized with Proteobacteria
during the first weeks of life during which early immune protection or tolerance might develop, may not be able to modulate an adaptive immunological response to a subsequent bloom of Proteobacteria
, and instead develop intestinal pathology consistent with NEC.
A recently published study
reported that, using similar sequencing techniques, less than 10% of healthy infants carried Enterobacteria
until the sixth week of life when a bloom occurred. Our study went further as we detected specific OTUs that increased in NEC infants during the week prior to diagnosis. Most of these OTUs grouped to the Enterobacteriaceae
, and represented both known and unknown species. An OTU matching S. epidermidis
, a common skin commensal, was also exclusively detected in infants with NEC.
We could not confirm a report by others that suggested a contribution of clostridia species and their toxins to NEC.
We did not observe an association of NEC risk with the presence of Citrobacter
like sequences or the frequency of Enterococc
usas suggested in preliminary studies.
However, in the current study we have sufficient power for detecting differences between groups in any OUT's that contributed at least 0.5% to the overall microbiota in the majority of samples from cases or controls.
Our observations regarding any of the OUT's that were more frequently detected in cases should be interpreted with caution. We initially compared distributions of more than a thousand OUT's. Even after removal of rare OUT's we retained many hundreds of OUT's in our analysis. Due to the exploratory character of our study we did not correct for multiple analyses, although we did lower the significance level to p<0.01. Thus, we cannot exclude the possibility that some of our findings did occur by chance. Furthermore, as we studied microbiota development only in preterm infants born in Florida, our findings cannot necessarily be generalized to other populations.
Among the many OTUs that we evaluated we did detect an increase in the numbers of a particular OTU that matched closest to the Enterobacteriaceae
. When we searched Genbank for the closest match using a blast search, we did not detect any sequence that matched this OTU by more than 97%. This is an intriguing observation because the Enterobacteriaceae
family is particularly well-studied and contains many established enteric pathogens that harbor a variety of known virulence factors. Our finding is consistent with the hypothesis that a novel pathogen might be an important etiologic factor in at least a subset of infants with NEC. One line of evidence that provides support for the notion that this organism belongs to a class of microbes that has special pathogenic characteristics involves the lipid A moiety of the cell wall LPS, which is known to differ between organisms. Animals use the TLR4 receptor mechanism to recognize the lipid A moiety of LPS. LPS recognition and pro-inflammatory signaling by TLR4 occurs only when lipid A has a certain structure; it contains both phosphates and is a hexaacyl.
In the Bacteroidetes
that sometimes dominate the gut microbiota in adults, the LPS is pentacylated, making it a less efficient activator of the TLR4 mechanism leading to decreased immunogenicity.
However in Proteobacteria
, the lipid A is hexacylated, making it a potent agonist for the TLR4 system and thus more immunogenic. During times of health, the immunogenic properties of the LPS of Proteobacteria
are muted by the overwhelming predominance of Bacteroidetes
. However, during inflammation (as occurs in diseases such as NEC), there is a relative decrease in the presence of these less immunogenic bacteria and a relative increase in Proteobacteria
, as shown by our results.
In addition to specific OTUs that appeared to be associated with NEC we also detected a bloom in Proteobacteria unique to the NEC infants. Our observations require confirmation in further studies and with different methodologies. Such studies are currently ongoing at our institution and elsewhere as part of the Human Microbiome Project. We are hopeful that a better understanding of NEC etiology from such studies might be rapidly translated into better diagnostic tools and prophylactic interventions that will meaningfully reduce the incidence and severity of NEC in preterm infants.