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Redefining the Role of Intestinal Microbes in the Pathogenesis of Necrotizing Enterocolitis Michael J. Morowitz, Valeriy Poroyko, Michael Caplan, John Alverdy and Donald C. Liu Pediatrics 2010;125;777; originally published online March 22, 2010; DOI: 10.1542/peds.2009-3149

The online version of this article, along with updated information and services, is located on the World Wide Web at:
http://pediatrics.aappublications.org/content/125/4/777.full.html

PEDIATRICS is the official journal of the American Academy of Pediatrics. A monthly publication, it has been published continuously since 1948. PEDIATRICS is owned, published, and trademarked by the American Academy of Pediatrics, 141 Northwest Point Boulevard, Elk Grove Village, Illinois, 60007. Copyright © 2010 by the American Academy of Pediatrics. All rights reserved. Print ISSN: 0031-4005. Online ISSN: 1098-4275.

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Redefining the Role of Intestinal Microbes in the Pathogenesis of Necrotizing Enterocolitis
AUTHORS: Michael J. Morowitz, MD,a,b Valeriy Poroyko, PhD,a Michael Caplan, MD,c John Alverdy, MD,a and Donald C. Liu, MD, PhDa,b
Departments of aSurgery and bPediatrics, University of Chicago Pritzker School of Medicine, Chicago, Illinois; and cDepartment of Pediatrics, NorthShore University HealthSystem, Evanston, Illinois KEY WORDS enterocolitis, necrotizing, bacteria, Archaea, viruses, enteral nutrition, infant, newborn, premature, humans ABBREVIATIONS NEC—necrotizing enterocolitis VLBW—very low birth weight TLR—Toll-like receptor iNOS—inducible nitric oxide synthetase www.pediatrics.org/cgi/doi/10.1542/peds.2009-3149 doi:10.1542/peds.2009-3149 Accepted for publication Jan 13, 2010 Address correspondence to Michael J. Morowitz, MD, Department of Surgery, University of Chicago Pritzker School of Medicine, 5841 S Maryland Ave, MC 4062, Chicago, IL 60637. E-mail: [email protected] PEDIATRICS (ISSN Numbers: Print, 0031-4005; Online, 1098-4275). Copyright © 2010 by the American Academy of Pediatrics FINANCIAL DISCLOSURE: The authors have indicated they have no financial relationships relevant to this article to disclose. Funded by the National Institutes of Health (NIH).

abstract
Neonatal necrotizing enterocolitis (NEC) remains an important cause of morbidity and mortality among very low birth weight infants. It has long been suspected that intestinal microbes contribute to the pathogenesis of NEC, but the details of this relationship remain poorly understood. Recent advances in molecular biology and enteric microbiology have improved our ability to characterize intestinal microbes from infants with NEC and from healthy unaffected newborns. The lack of diversity within the neonatal intestine makes it possible to study gut microbial communities at a high level of resolution not currently possible in corresponding studies of the adult intestinal tract. Here, we summarize clinical and laboratory evidence that supports the hypothesis that NEC is a microbe-mediated disorder. In addition, we detail recent technologic advances that may be harnessed to perform highthroughput, comprehensive studies of the gut microbes of very low birth weight infants. Methods for characterizing microbial genotype are discussed, as are methods of identifying patterns of gene expression, protein expression, and metabolite production. Application of these technologies to biological samples from affected and unaffected newborns may lead to advances in the care of infants who are at risk for the unabated problem of NEC. Pediatrics 2010;125:777–785

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777

Neonatal necrotizing enterocolitis (NEC) remains an important cause of morbidity and mortality among very low birth weight (VLBW) infants. Paradoxically, although nearly every review article about NEC refers to the importance of gut bacteria in disease development,1,2 precious little is known about the molecular mechanisms by which microbes contribute to NEC pathogenesis. In the last decade, revolutionary advances in molecular biology and microbiology have dramatically improved our understanding of the microbes present within the human gastrointestinal tract. It is now well established that intestinal microbes serve vital functions during normal development and health maintenance and that they also contribute to pathogenesis of human disease.3–5 Intestinal microbes (ie, bacteria and Archaea, and their associated viruses) are currently the focus of many investigations, the aim of which is to define normal and abnormal enteric microbiology. This renaissance in enteric microbiology offers renewed opportunities to better understand the pathophysiology of NEC. Here, we summarize what is known about the role of microbes in NEC pathogenesis and describe how technologic advances may be harnessed to advance the field.

tion that NEC is mediated, at least in part, by intestinal microbes has continued to mount. This evidence includes the common findings of bacteremia8–10 and endotoxinemia11,12 in affected neonates and the pathognomonic imaging finding of pneumatosis intestinalis, which likely represents submucosal gas produced by bacterial fermentation.13 Moreover, occasional “clusters” of cases linked to causative pathogens have been reported, although the identity of the responsible organisms has varied among outbreaks and across institutions.14 The efficacy of broad-spectrum antibiotic therapy in many cases of NEC and the apparent efficacy of probiotic administration in some clinical trials15 have provided further indirect evidence that manipulation of the intestinal microbial community can affect outcomes in infants with NEC. Given the strength of the evidence (albeit often circumstantial) that microbes contribute to the pathogenesis of NEC, clinicians and researchers have been searching for more than 3 decades to identify organisms that are causally linked with the disease. Until recently, such efforts have been restricted to culture-based investigations of blood, peritoneal fluid, or intestinal contents of patients with NEC or control populations. The results of these investigations have been highly variable and, taken as a whole, inconclusive. Three fascinating reports in The Lancet 30 years ago demonstrated the presence of clostridial species in samples from infants in NEC outbreaks.16–18 Since that time, results of several other investigations have supported a role for clostridial species (including Clostridium perfringens, Clostridium butyricum, and Clostridium neonatale) in NEC pathogenesis.19–21 It has been suggested (but never proven) that the gas-forming ability of clostridia accounts for pneu-

matosis intestinalis in affected patients.22 Results of important work from Kosloske and Ulrich23 indicated that the disease is generally more severe when C perfringens can be cultured from the blood or peritoneal fluid of an affected patient. Nevertheless, others have convincingly demonstrated that the prevalence of colonization with clostridial species (including Clostridium difficile) does not differ between affected infants and healthy controls.24–27 In general, with respect to both clostridial and nonclostridial organisms, microbial species identified in cultures from patients with NEC have not been significantly different from species generally considered “normal” within the gastrointestinal tract of hospitalized patients. A partial list of organisms associated in various reports with NEC (Table 1) includes Klebsiella pneumoniae, Escherichia coli, Enterobacter spp, Pseudomonas spp, C difficile, and Staphylococcus epidermidis.22,24,28–30 Because these organisms are also commonly found among patients without NEC, none of them fulfill Koch’s postulates31 that define causal relationships between microbes and disease pathogenesis. The inconsistency of these findings over several decades strongly suggests that no single causative pathogen is responsible for NEC pathogenesis. However, it is still possible that causative organisms do actually exist and

WHY NEC IS CONSIDERED A MICROBE-MEDIATED DISEASE
Clinical Evidence More than 30 years ago, in 2 classic articles, Sa ´ ntulli et al6,7 identified bacterial colonization as 1 of 3 critical factors (in addition to mucosal injury and initiation of enteral feedings) that predispose newborn infants to NEC. This conclusion was based on experience caring for Ͼ100 infants with the disease at the Infants’ Hospital in New York between 1955 and 1979. Since that time, evidence supporting the no778 MOROWITZ et al

TABLE 1 Partial List of Microbial Species That
Have Been Linked to NEC Pathogenesis
Bacteria Clostridium perfringens Clostridium butyricum Clostridium neonatale Clostridium difficile Klebsiella pneumoniae Escherichia coli Cronobacter sakazakii Staphylococcus epidermidis Pseudomonas aeruginosa Viruses Coronavirus Coxsackie B2 virus Rotavirus Adenovirus Torovirus Astrovirus Echovirus 22

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that insufficiencies in present cultivation techniques have prevented their proper identification. Although far more attention has been paid recently to intestinal bacteria, the distinct possibility remains that enteric viruses play a critical role in NEC pathogenesis. Much less is known about the normal viral load or potential viral pathogens in the newborn intestine, partly because viruses are more difficult to study than bacteria. However, outbreaks of the disease over the past 4 decades have provided unique opportunities to make associations between NEC and viruses. More than 2 decades ago, a series of remarkable studies demonstrated the presence of a novel coronavirus within fecal samples and resected intestinal segments from infants with NEC.32,33 Subsequently, results of other studies that used a variety of experimental approaches have linked the disease at various times and locations to other viruses including coxsackie B2 virus, rotavirus, adenovirus, torovirus, astrovirus, and echovirus 22.34–38 Few if any studies have been conducted to systematically evaluate the contribution of viruses to NEC pathogenesis, partly because of the inherent difficulties in studying viruses in clinical settings. Thus, the role of viruses in NEC pathogenesis remains uncertain at present. Laboratory Evidence Extensive work with animal models of NEC has repeatedly confirmed the importance of intestinal bacteria in disease pathogenesis. Barlow et al used a newborn rat model to demonstrate that animals with NEC had a marked overgrowth of Gram-negative organisms in the stool and blood relative to control animals without disease.39 In 1986, Kosloske and co-workers40 compared NEC development in germ-free and wild-type newborn rats and deterPEDIATRICS Volume 125, Number 4, April 2010

mined bacteria to be the most critical factor to determine disease pathogenesis. In 2006, Jilling et al41 reported that sanitization of feeding catheters between enteral feedings markedly diminished the incidence of disease in their well-validated rodent models of NEC. In numerous additional studies, utilizing quail42 and piglet models43,44 and the more common rodent models,45,46 the importance of bacteria in NEC pathogenesis has been confirmed. Collectively, these studies defined gut bacteria as necessary for disease, but they did not clarify how and why they are necessary. More recently, laboratory investigations of NEC have begun to elucidate putative molecular mechanisms by which gut microbes contribute to the development of disease. The most promising advances thus far relate to the importance of Toll-like receptors (TLRs) in NEC pathophysiology. TLRs are integral glycoproteins that allow cells of the innate immune system to recognize highly conserved molecules derived from microbes such as lipopolysaccharides, bacterial flagellin proteins, or microbial nucleic acids.47 In recent years, a pair of important publications detailed the relevance of TLR4 activation in NEC pathogenesis. Jilling et al41 elegantly demonstrated that enterocyte expression of TLR4 increases after birth in animals with risk factors for NEC (ie, formula feeding and environmental stress), whereas TLR4 expression in enterocytes from healthy mother-fed controls gradually decreases during the first 3 days of life. It is important to note that they also demonstrated that TLR4-null mice were protected from disease relative to wild-type animals in the mouse model of NEC. Hackam and coworkers48 soon thereafter confirmed some of these findings and documented increased TLR4 expression in intestinal epithelium resected from

human infants with NEC. They further demonstrated that the role of TLR4 in disease formation relates to enterocyte injury and repair. It is interesting to note that additional data have suggested that TLR4 expression can be induced by platelet-activating factor, a molecule that has been previously implicated in the pathogenesis of mucosal injury in NEC.49 Other notable work has identified the importance of inducible nitric oxide synthetase (iNOS), which is upregulated in the presence of microbes, in the development of experimental NEC.43,50,51 In the setting of inflammation, increased iNOS activity is believed to generate toxic intermediates of nitric oxide that can compromise mucosal integrity. Future work must elucidate the mechanisms by which bacteria activate TLR4 and iNOS pathways (among others) in the setting of NEC and must determine if the so-called normal commensal bacteria, or invading pathogens, are primarily responsible for activating these pathways during disease pathogenesis.

CULTURE-INDEPENDENT STUDIES OF GUT MICROBES IN LOW BIRTH WEIGHT INFANTS
Over the past decade, knowledge of microbes present within the human gastrointestinal tract has expanded dramatically with the advent of molecular culture-independent techniques in microbiology. Such interrogations have indicated that perhaps only 20% of enteric bacteria can be grown under standard laboratory conditions.52 Most frequently, such methods have employed polymerase chain reaction amplification of conserved marker genes such as the gene encoding the 16S ribosomal RNA subunit.53–55 This gene is present in all prokaryotic cells; genomic sequences for this gene are nearly identical in different bacterial species, except within hypervariable
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regions. Amplification and sequencing of these hypervariable regions can provide a molecular “fingerprint” that enables accurate identification of a particular species within a complex biological sample such as fecal matter. The body of literature in which 16Sbased methods have been used to characterize mammalian gut flora in both healthy and disease states has grown exponentially in the past 10 years.56–58 In parallel with these developments, a number of studies have emerged in which culture-independent molecular techniques were used to examine the fecal flora of both term and preterm infants. Such studies have led to 2 important conclusions with relevance to NEC. First, the number of species present within the newborn intestine is low. Whereas estimates of the bacterial load in the adult intestine range from 100 to 1000 species,59 studies published to date have indicated that the distal gastrointestinal tract of VLBW infants during the first few weeks of life may contain fewer than 20 bacterial species.60–62 Second, profiles of gut microbes in the first months of life are characterized by a remarkable interindividual variability.27,60,62,63 However, by 6 or 12 months of age, variation between individuals is far less pronounced. What these results reinforce is that the gut microbial communities of newborns are tenuous and unstable relative to older children and adults. It is logical, therefore, to pursue the still unproven hypothesis that changes within these fragile microbial communities correlate with the onset of microbemediated diseases such as NEC. Thus far, 4 small studies have characterized 16S ribosomal RNA gene sequences from gut bacteria of human infants with NEC. Millar et al64 published the first such study in 1996 and did not identify significant differences
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in the fecal flora of infants with and without NEC. However, a limitation of their study was that each of the samples obtained from infants with NEC (with 1 exception) was obtained after onset of disease and initiation of antibiotics. The second article in this group, published by de la Cochetiere et al, compared microbial species present within fecal samples during the first 2 weeks of life in 3 patients with NEC and 9 matched controls.65 The samples from the infants with NEC were collected prospectively in this study. It was intriguing that they found that C perfringens was present before disease in samples from each of the 3 infants with NEC, whereas it could not be demonstrated in any control samples. The obvious limitation of this study was the small size, yet the results are consistent with the notion that early colonization by C perfringens predisposes to later development of NEC. Wang et al66 completed the largest of the 4 cultureindependent studies of the microbiology of human NEC. In their study, 16S gene sequence analysis was used to characterize the fecal flora of 10 preterm infants with NEC and 10 matched controls. These authors used recently developed bioinformatic techniques in conjunction with 16S profiling to demonstrate that NEC samples clearly clustered separately from non-NEC samples and that NEC samples were characterized by diminished overall microbial diversity and an abundance of gammaproteobacteria species. However, as was the case in the Millar study, no samples in this study were collected before the onset of disease; as a result, it is impossible to eliminate the possibility that these findings were secondary to administration of antibiotics or, perhaps, secondary to the mucosal inflammation inherent to the primary disease process.

Most recently, Mshvildadze et al27 published results of the first study to use a high-throughput technology (454 pyrosequencing) to characterize 16S gene sequences within fecal samples from neonates. These authors conducted a thorough phylogenetic analysis of fecal microbes from 6 VLBW infants with NEC and/or sepsis and from 6 matched controls. Samples from affected infants were obtained approximately 1 week before the onset of disease. No significant differences were observed in the overall structure of microbial communities from infants with and without NEC. However, a few intriguing findings were noted, including increased abundance of Enterococcus and Citrobacter gene sequences from affected patients. Clearly, a prospective, large-scale, culture-independent study of gut microbes is needed to build on these groundbreaking preliminary studies to definitively determine if onset of NEC is associated with alterations in the structure of gut bacterial communities.

APPLICATION OF HIGHTHROUGHPUT TECHNOLOGIES TO STUDY INTESTINAL MICROBES
The dramatic new opportunities now available to study intestinal microbes in the context of NEC have largely resulted from 2 developments over the past decade. The first, as discussed above, was the transition from culture-based to culture-independent interrogations. The second important development was the introduction of extraordinary high-throughput technologies with clear relevance to enteric microbiology. Recently developed sequencing platforms (eg, GS FLX [Roche, Indianapolis, IN] and ABI SOLiD [Applied Biosystems Inc, Foster City, CA]) are making it possible to sequence massive amounts of DNA or RNA isolated from microbial specimens67; similarly important advances in mass spectrometry have enabled

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FIGURE 1
DNA-based investigations of microbial community structure.

characterizations of thousands of microbial proteins or metabolic byproducts within biological specimens.68 Although it was previously unfeasible to screen all or nearly all DNA, RNA, or proteins within biological samples from multiple patients, such investigations are now feasible and, in fact, underway. Metagenomics Accumulating experience with the molecular characterization of intestinal microbes has clearly demonstrated the value of sequencing all microbial DNA within the intestine and not solely the gene sequences of marker genes such as 16S69 (Fig 1). Studies that aim to make taxonomic identifications of gut microbes overlook the reality that 2 strains of a given bacterial species may differ in DNA content by as much as 25%.70 For example, a 16S-based study may identify the presence of an E coli species but would not provide any information about whether that particular strain of E coli is predicted (on the basis of DNA content) to be a harmless commensal or harmful pathogen. Such DNA variation, termed genome plasticity, can have important consequences for the clinical phenotype of an organism. Therefore, 16S-based studies fail to provide predictions of bacterial community function that are made possible by sequencing the entire intestinal “metagenome.” The metagenome (or “community genome”) is defined as the combined genomes of all species within a mixed miPEDIATRICS Volume 125, Number 4, April 2010

crobial population, and sequencing metagenomes was not technically feasible before the recent advances in sequencing technology.69 The initial metagenomic studies of the distal human intestine were path-breaking because they proved that the gut microbiome encodes for a surprisingly diverse array of metabolic processes71,72; in fact, these studies proved that gut microbes are essential for degrading specific components of the human diet. In addition, metagenomic sequencing has identified the importance of DNA sequences related to microbial virulence and horizontal gene transfer,73,74 both of which could be important to NEC pathogenesis. Successfully characterizing all microbial DNA within biological samples is substantially more difficult when samples are complex. Because the neonatal intestinal tract contains far fewer species than the adult intestinal tract, a metagenomic approach offers the possibility of more fully characterizing the gut microbial metagenomes of individual patients at risk for NEC. As the cost of high-throughput sequencing continues to decrease, it should become feasible to conduct an adequately powered comparison of metagenomes from infants with and without NEC. Metagenomics offers particular promise for investigations of viruses within the gastrointestinal tract. In contrast to marker genes such as 16S that are present in all bacterial species, there are no RNA or DNA sequences shared

by all viruses that can be used for easy culture-independent species identification.75 However, if all viral DNAs and RNAs within a biological sample are sequenced, then the diversity and complexity of the viral community can be characterized accurately. Such an approach has been used in limited instances to demonstrate that a large number of viruses within the gastrointestinal tract are actually bacteriophages that infect gut bacteria.76 A recent study demonstrated that, whereas meconium from a newborn infant did not contain viral particles, samples obtained 1 week later from the same infant contained over 108 viral particles per gram of feces.77 Another study used a metagenomic approach to identify novel viruses present within stool samples from pediatric patients with diarrhea.78 The clinical relevance of these findings is not yet known. Functional Characterizations of Intestinal Microbial Communities DNA-based investigations can delineate the genetic potential of a gut microbial community. However, it is well recognized that the presence of a particular microbial gene within the gut microbiome by no means indicates that the gene in question will be expressed. For example, documenting the presence of a gene encoding a protein involved in virulence or antibiotic resistance indicates that the gene might be expressed without indicating whether the gene actually is expressed in a given biological sample. As such, there is great interest in developing technologies to assess the phenotype of microbial communities. Three general lines of inquiry are now being applied to evaluate the function of naturally occurring microbial communities such as those found in the gastrointestinal tract (Fig 2). Each of these approaches is routinely used
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FIGURE 2
High-throughput investigations of microbial community function.

(eg, measurement of gene expression) to characterize behavior of bacterial monocultures under tightly controlled conditions in the laboratory. However, these tasks are made considerably more difficult when applying such technologies to study naturally occurring biological samples that contain unknown and often complex mixtures of microbial species. Metatranscriptomics, or “community transcriptomics,” seeks to identify the genes that are expressed by all species present within a complex specimen such as a fecal sample. This approach has recently been used to detail community gene expression in samples from ocean water and soil79–81; however, no published study has thus far detailed community gene expression within the intestinal tract. Metaproteomics, or “community proteomics,” is similarly the study of proteins collectively expressed within microbial communities. Particularly when corresponding metagenomic data sets are available, mass-spectrometry– based proteomic analysis has enabled the generation and subsequent validation of fascinating hypotheses regarding function of previously unrecognized proteins.82,83 Recently, 2 publications indicated that this approach is feasible in the study of gut microbes in humans.84,85 Finally, metabolomics is the study of low molecular weight microbial metabolites within biological samples. Identification of metabolites within a gut microbial community arguably serves as the most accurate measure of microbial function by unambiguously measuring
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the end products of gene and protein expression.86,87 Each of these highthroughput approaches to characterizing gut microbial communities has technical and theoretical advantages and disadvantages, and it has not yet been proven that functional studies of fecal microbes are informative with regard to microbial function elsewhere in the gastrointestinal tract. Although these approaches have not yet been exploited to study the microbiology of NEC, the simplicity of the juvenile intestinal tract may allow for successful completion of such studies in coming years.

between the gut microbial communities of milk-fed and formula-fed infants. To date, however, the results of studies that used culture-based techniques and 16S-based molecular profiling do not support a firm conclusion regarding differences between these 2 groups.27,63,90 However, highthroughput molecular investigations of this subject are only beginning to emerge and are likely to identify such differences. We recently completed a metabolomic analysis of fecal samples from milkfed and formula-fed premature infants by using a mass-spectrometry– based platform (V.P. and M.J.M., unpublished data). We identified a total of 174 chemical compounds within these samples, and the distribution of chemicals within these samples was sharply affected by diet. Principal component analysis of the data sets demonstrated that samples from milk-fed and formulafed infants clustered tightly together. These results indicate that the active metabolic pathways used by microbes within the intestinal tracts of milk-fed and formula-fed infants are distinct. The application of other high-throughput technologies will help further define the impact of diet on the gut microbial communities of newborn infants. Probiotics and the Intestinal Microbial Community Multiple clinical studies have been performed to determine if the incidence of NEC can be reduced by the administration of probiotics (defined as dietary supplements of live microorganisms that are derived from the human intestinal tract and that have putative health benefits). The hypothesis that probiotics might help prevent NEC stems from the observation that the prevalence of probiotic genera such as Bifidobacterium and Lactobacillus is lower within the gastrointestinal tracts of hospitalized preterm infants than in otherwise

UNRESOLVED QUESTIONS RELATED TO INTESTINAL MICROBES AND NEC PATHOGENESIS
Diet and the Intestinal Microbial Community An important lesson from the pathbreaking research over the past decade has been that nutrition, gut microbial function, and energy metabolism are tightly intertwined.4,88 This has particular relevance to VLBW infants, because the incidence of microbe-mediated disease such as NEC and late-onset sepsis is significantly higher in formula-fed infants.89 A critical and unresolved issue is how gut microbial communities differ in infants who receive artificial formula and those who receive maternal milk. Because different dietary sources can be viewed as distinct “growth media” for intestinal microbes, it makes sense intuitively that differences must exist

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healthy term infants.15 A recent metaanalysis concluded that enteral supplementation of probiotics does reduce the risk of developing NEC for low birth weight infants.91 Although numerous questions remain regarding safety, dosage, and the specific species used in different formulations, the prospect of a new strategy to prevent onset of NEC is exciting. Nonetheless, there are several important points to be made about probiotics and NEC in the context of emerging technologies to characterize intestinal microbes. First, the purported deficiency of “beneficial” gut bacteria in premature infants is based on older, culture-based studies and, thus, should be viewed with caution. Ongoing studies to document colonization patterns with molecular techniques will be particularly valuable in confirming or refuting such long-held beliefs. Second, no culture-independent studies have demonstrated that infants with NEC have lower counts of probiotic species than matched controls without NEC. Proving such a conclusion REFERENCES
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(which may be possible with studies now in progress) would greatly strengthen the rationale for using probiotics. However, if the pathogenesis of NEC is truly related to a deficiency of favorable bacteria, and if this deficiency is shared by all premature infants, one must wonder why more than 90% of preterm infants lack those desirable bugs but do not develop the disease. Likewise, it must be considered whether the benefits outweigh the risks in administering live bacterial supplements to all patients at risk. The difficulty, of course, lies in prospectively identifying which patients are at highest risk for developing the disease. At present, there is no effective method to ascertain which 5% to 10% of VLBW infants will develop the disease. Theoretically, there may be some characteristics that distinguish the gut microbes of patients who eventually develop NEC from those who do not. If this is true, it may be feasible to give probiotics to a select group of patients at high risk rather than to all patients.

CONCLUSIONS
Recent advances in the study of intestinal microbes have demonstrated that the microbial ecosystem within the human gastrointestinal tract is exceedingly complex. However, the advent of new high-throughput technologies has led to promising new opportunities to study the contribution of gut microbes to NEC pathogenesis. This remains a highly relevant line of inquiry with regard to the care of low birth weight infants given that the burden of the disease remains high and because the results of numerous studies have suggested that microbes contribute to disease pathogenesis. The gut microbial ecosystems of premature infants are uniquely amenable to the use of high-throughput technologies such as metagenomics because of the lack of microbial diversity in the newborn intestine. Application of these technologies to biological samples from affected and unaffected newborns may lead to advances in the care of low birth weight infants who are at risk for the unabated problem of NEC.

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PEDIATRICS Volume 125, Number 4, April 2010

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Redefining the Role of Intestinal Microbes in the Pathogenesis of Necrotizing Enterocolitis Michael J. Morowitz, Valeriy Poroyko, Michael Caplan, John Alverdy and Donald C. Liu Pediatrics 2010;125;777; originally published online March 22, 2010; DOI: 10.1542/peds.2009-3149
Updated Information & Services including high resolution figures, can be found at: http://pediatrics.aappublications.org/content/125/4/777.full.ht ml This article cites 90 articles, 31 of which can be accessed free at: http://pediatrics.aappublications.org/content/125/4/777.full.ht ml#ref-list-1 This article has been cited by 8 HighWire-hosted articles: http://pediatrics.aappublications.org/content/125/4/777.full.ht ml#related-urls Information about reproducing this article in parts (figures, tables) or in its entirety can be found online at: http://pediatrics.aappublications.org/site/misc/Permissions.xht ml Information about ordering reprints can be found online: http://pediatrics.aappublications.org/site/misc/reprints.xhtml

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PEDIATRICS is the official journal of the American Academy of Pediatrics. A monthly publication, it has been published continuously since 1948. PEDIATRICS is owned, published, and trademarked by the American Academy of Pediatrics, 141 Northwest Point Boulevard, Elk Grove Village, Illinois, 60007. Copyright © 2010 by the American Academy of Pediatrics. All rights reserved. Print ISSN: 0031-4005. Online ISSN: 1098-4275.

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