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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

 

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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, Illi nois, 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

abstract

Departments of   a Surgery and  b Pediatrics, University of Chicago  Pritzker School of Medicine, Chicago, Illinois; and  c Department  of Pediatrics, NorthShore University HealthSystem, Evanston, Illinois 

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 high throughput, 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  2010;125:777–785

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 DISCLOSURE::  The authors have indicated they have  no financial relationships relevant to this article to disclose. Funded by the National Institutes of Health (NIH).

PEDIATRICS PEDIAT RICS Volume 125, Number 4, April 2010

 

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Neonatal necrotizing enterocolitis (NEC) remains an important cause of morbidity and mortality among very low birth weight (VLBW) infants. Paradoxically, cal ly, although nearly every review article about NEC refers to the impor tance of gut bacte bacteria ria in dise disease ase de1,2 velopment, precious little is known

 tion that NEC is mediated, at least in part, by intestinal microbes has con tinued to mount. This evidence includes clu des the com common mon findings findings of bac bac-8–10 11,12  teremia and endot endotoxine oxinemia mia in affected neonates and the pathognomonic imaging finding of pneumatosis intestinal intes tinalis, is, which likely repre represents sents

matosiss int matosi intest estina inalis lis in aff affect ected ed pa tients.22 Res Result ultss of imp impor ortan tantt wor work k 23 from Kosloske and Ulrich indicated  that the disease is generally more severe when  C perfringens   can be cul tured from the blood or peritoneal fluid of an affected patient. Nevertheless, others have convincingly demon-

about the molecular mechanisms by which whic h mic microbe robess cont contribu ribute te to NEC pathogenesis. In the last decade, revolutionaryy advances in molecular biolutionar ology and microbiology have dramatically improved our understanding of   the micro microbes bes pres present ent withi within n the human gastrointestinal tract. It is now well established that intestinal microbe cr obess ser serve ve vi vital tal fun funct ctio ions ns dur dur-ing normal development and health maintenance and that they also con tribute  tribu te to path pathogen ogenesis esis of huma human n

submucosal gas produ submucosal produced ced by bacte bacterial rial fermentation.13 Moreover, occasional “clusters” of cases linked to causative pathog pat hogens ens hav have e bee been n rep report orted, ed, al though the identity of the responsible orga or gani nism smss ha hass va vari ried ed am amon ong g ou outtbreaks and across institutions.14 The efficacy of broad-spectrum antibiotic  therapy in many cases of NEC and the apparent appar ent effica efficacy cy of probi probiotic otic admin adminisis tration in some clinical trials15 have provided provi ded furth further er indir indirect ect evide evidence nce that manipulation of the intestinal micro-

strate str ated d tha thatt the pre preval valenc ence e of col coloni onizaza tion with clostridial species (including Clostridium difficile ) doe doess not differ differ between affected infants and healthy controls.24–27

bial community can affect outcomes in infants with NEC.

organismss assoc organism associated iated in vari various ous reports with NEC (Table 1) includes  Kleb-  siella siel la pneum pneumoniae  oniae ,   Escherichia Escherichia coli ,   spp,  Pseudomonas   spp, Enterobacter  spp, C difficile , and  Staphylococcus epider-  midis .22,24,28–30 Bec Becaus ause e the these se or organ gan-isms are also commonly found among patients without NEC, none of them fulfill Koch’s postulates31  that defin define e causal caus al rela relatio tionshi nships ps betw between een mi mi-crobes and disease pathogenesis. The inconsist incon sistency ency of these findings over severa sev erall dec decade adess str strong ongly ly sug sugges gests ts  that no single causative pathogen is respon res ponsib sible le for NE NEC C pat pathog hogene enesis sis.. However, it is still possible that causative organisms do actually exist and

In general, with respect to both clos tridial and nonclostridial organisms, microb mic robial ial spe specie ciess ide identi ntified fied in cul cul- tures from patients with NEC have not been significantly different from species gener generally ally cons considere idered d “norm “normal” al” within wit hin the gas gastro troint intest estina inall tra tract ct of  hospitalized patients. A partial list of 

3–5

disease. Intesti Inte stinal nal mic microbe robess (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 understa under stand nd the path pathophy ophysio siology logy of  NEC. NE C. He Here, re, we sum summa mari rize ze wha whatt is known about the role of microbes in NEC pathogenesis and describe how  technolog  tech nologic ic advan advances ces may be harnessed to advance the field.

Given the strength of the evidence (albeitt oft bei often en cir circum cumsta stanti ntial) al) tha thatt microbes contribute to the pathogenesis of NEC NEC,, cli clinic nician ianss and res resear earche chers rs have been searching for more than 3 decades to identify organisms that are causally linked with the disease. Until recent rec ently, ly, suc such h eff effort ortss hav have e bee been n restricted stri cted to cultu culture-ba re-based sed inves investigatiga tions of blood, peritoneal fluid, or in testinal contents of patients with NEC

More than 30 years ago, in 2 classic articles, Sa´ ntulli et al 6,7 identified bac terial colonization as 1 of 3 critical fac tors (in addition to mucosal injury and initiation of enteral feedings) that predispose newborn infants to NEC. This conclusion was based on experience caring for 100 infants with the dis-

or control populations. The results of   these investigations have been highly variable and, taken as a whole, inconclusive. clusi ve. Three fascinating fascinating repor reports ts in The Lancet  30  30 years ago demonstrated  the presence of clostridial species in samp sa mple less fr from om in infa fant ntss in NE NEC C ou outt16–18 breaks. Since that time, results of  several other investigations have supported porte d a role for clos clostridi tridial al speci species es (including   Clostridi Clostridium um perfrin perfringens  gens , Clostridiu Clost ridium m butyri butyricum  cum , and   Clostrid-  ium neo neonat natale  ale ) in NE NEC C pa path thog ogen enee-

ease at the Inf ease Infant ants’ s’ Hos Hospit pital al in New York Yor k bet betwee ween n 195 1955 5 and 197 1979. 9. Sin Since ce  that time, evidence supporting the non o-

sis. It ha hass be been en su sugg gges este ted d (b (but ut never nev er pro proven ven)) tha thatt the gas gas-fo -formi rming ng ability of clostridia accounts for pneu-

WHY NEC IS CONSIDERED A MICROBE-MEDIATED DISEASE Clinical Evidence

19–21

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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   

Viruses        

Cronobacter sakazakii    Staphylococcus epidermidis  Pseudomonas aeruginosa 

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Coronavirus Coxsackie B2 virus Rotavirus Adenovirus Torovirus Astrovirus Echovirus 22

 

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 that insufficiencies in present cultiva tion techniques have prevented their proper identification. Although far more attention has been paid pai d rec recent ently ly to int intest estina inall bac bacter teria, ia, the distinct disti nct poss possibili ibility ty rema remains ins that en teric viruses play a critical role in NEC pathog pat hogene enesis sis.. Muc Much h les lesss is kno known wn about the normal viral load or poten tial viral pathogens in the newborn in testine, partly because viruses are more difficult to study than bacteria. Howeve How ever, r, out outbre breaks aks of the dis diseas ease e over the past 4 decades have provided unique opportunities to make associa tions between NEC and viruses. More  than 2 decades ago, a series of remarkable mark able studi studies es demons demonstrat trated ed the presen pre sence ce of a nov novel el cor corona onavir virus us wit within hin fecal samples and resected intestinal segmen seg ments ts fro from m inf infant antss wit with h NEC NEC..32,33

mined bacteria to be the most critical factor to determine disease pathogenesis. In 2006, Jilling et al 41 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,

human infants with NEC. They further demonstrated that the role of TLR4 in disease disea se form formatio ation n rela relates tes to enter enteroocyte 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 im-

utilizing quail42 and piglet models43,44 and the more common rodent models,45,46  the importance of bacteria in NEC pat pathog hogene enesis sis has bee been n con confirm firmed. ed. Collectively, these studies defined gut bacteria as necessary for disease, but  they did not clarify how and why they are necessary.

plicated in the pathogenesis of mucosal injury in NEC.49

More recent recently, ly, labor laborator atoryy inves investigatiga tions of NEC have begun to elucidate putati put ative ve mol molecu ecular lar mec mechan hanism ismss by which gut microbes contribute to the devel dev elop opme ment nt of di dise seas ase. e. Th The e mo most st

Other notable work has identified the importanc impo rtance e of induci inducible ble nitri nitricc oxide synthetas synt hetase e (iNOS (iNOS), ), which is upreg upreguulated lat ed in the pre presen sence ce of mic microb robes, es, in the dev develo elopme pment nt of exp experi erimen mental tal 43,50,51 NEC. In the setting of inflamma tion, increased iNOS activity is believed  to generate toxic intermediates of  nitric oxide that can compromise mucosal integrity. Future work must elucidate cidat e the mechanisms mechanisms by which bac-

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, rotaviru rota virus, s, adenov adenovirus, irus, toro toroviru virus, s, as34–38  trovirus, and echovirus 22. Few if  any studies have been conducted to systemat syst ematicall icallyy evalu evaluate ate the contr contribuibu tion of viruses to NEC pathogenesis, partly because of the inherent difficul ties in studying viruses in clinical set tings. Thus, the role of viruses in NEC

promising advances thus far relate to  the importance of Toll-like receptors (TLRs)) in NEC patho (TLRs pathophysi physiology ology.. TLRs are integral glycoproteins that allow cells of the innate immune system to recognize highly conserved molecules derived from microbes such as lipopolysacch polys accharide arides, s, bacte bacterial rial flagel flagellin lin 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

 teria activate TLR4 and iNOS pathways (among others) in the setting of NEC and mus mustt det determ ermine ine if the soso-cal called led normal commensal bacteria, or invading pathogens, are primarily responsible for activating these pathways during disease pathogenesis.

pathogene pathog enesis sis rem remain ainss unc uncert ertain ain at present.

 that enterocyte expression of TLR4 increase cre asess aft after er bir birth th in ani animal malss wit with h ris risk k factor fac torss for NEC (ie (ie,, for formul mula a fee feedin ding g and envir environmen onmental tal stre stress), ss), where whereas as 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 mode dell of NEC. Hackam and coworkers48 soon thereafter confirmed

 trointestinal tract has expanded dramatically with the advent of molecular culture-independent techniques in microbiology. Such interrogations have indicated that perhaps only 20% of en teric bacteria can be grown under standard stan dard labor laborator atoryy condi conditions tions..52 Most freque fre quentl ntly, y, suc such h met method hodss hav have e employed polymerase chain reaction amplification of conserved marker genes such as the gene encoding the 16S ribosomal boso mal RNA subuni subunit. t.53–55 Thi Thiss gen gene e is pr pres esen entt in al alll pr prok okar aryo yoti ticc ce cell lls; s;

some of th some thes ese e fin findi ding ngss an and d do docu cu-mented increased TLR4 expression in intestinal intes tinal epith epithelium elium resec resected ted from

genomic sequences for this gene are nearly identical in different bacterial species, speci es, excep exceptt withi within n hyper hypervari variable able

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 newbor new born n rat mod model el to dem demons onstra trate te  that animals with NEC had a marked overgrow over growth th of GramGram-negat negative ive orga organnisms in the stool and blood relative to control animals without disease.39 In 40

1986, Kosloske and co-workers compared NEC development in germ-free and wild-type newborn rats and deter-

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CULTURE-INDEPENDENT STUDIES CULTURE-INDEPENDENT OF GUT MICROBES IN LOW BIRTH WEIGHT INFANTS Over the pas Over pastt dec decade ade,, kno knowle wledge dge of microbes present within the human gas-

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regions. Amplification and sequencing of the these se hyp hyperv ervari ariabl able e reg region ionss can provide a molecular “fingerprint” that enable ena bless acc accura urate te ide identi ntifica ficatio tion n of a particular species within a complex biological sample such as fecal matter. The body of literature in which 16Sbase ba sed d me meth thod odss ha have ve be been en us used ed to

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 excep exception) tion) was obtai obtained ned after onset of disease and initiation of antibiot bi otic ics. s. The se seco cond nd ar arti ticl cle e in th this is group,, publi group published shed by de la Cochet Cochetiere iere

Most recently, Mshvildadze et al27 publish li shed ed re resu sult ltss of th the e fir first st st stud udyy to us use ea high-throughput technology (454 pyrosequencing) to characterize 16S gene sequences within fecal samples from neonates. neona tes. These autho authors rs conduc conducted ted a  thorough phylogenetic analysis of fecal microbes from 6 VLBW infants with

characterize mammalian gut flora in both healthy and disease states has grown gro wn exp expone onenti ntiall allyy in the pas pastt 10 56–58 years. In parallel with these developments, a number num ber of stu studie diess hav have e eme emerge rged d 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. NE C. Fi Firs rst, t, th the e num numbe berr of sp spec ecie iess present within the newborn intestine

et al, com compar pared ed mic microb robial ial spe specie ciess present prese nt withi within n fecal samples during  the first 2 weeks of life in 3 patients with wit h NE NEC C and 9 mat matche ched d con contro trols. ls.65 The sam sample pless fro from m the inf infant antss wit with h NEC were coll collected ected prospectively prospectively in  this study. It was intriguing that they found that  C perfringens  was   was present before disease in samples from each of the 3 infants with NEC, whereas it coul co uld d no nott be de demo mons nstr trat ated ed in an anyy control contr ol sampl samples. es. The obvio obvious us limi limitata tion of this study was the small size,

NEC an NEC and/ d/or or se seps psis is an and d fr from om 6 ma matc tche hed d contro con trols. ls. Sam Sample pless fro from m aff affect ected ed 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  gene sequences from affected patients. Clearly, a prospec tive, large-scale, culture-independent

is low low.. Whe Wherea reass est estima imates tes of the bac bacteterial load in the adult intestine range from fro m 100 to 100 1000 0 spe specie cies, s,59 studies published to date have indicated that  the distal gastrointestina gastrointestinall tract of  VLBW VL BW in infa fant ntss du duri ring ng th the e fir first st fe few w weeks of life may contain fewer than 20 bacterial species.60–62 Second, profiles fil es of gu gutt mi micr crob obes es in th the e fir first st months of life are characterized by a remarkabl rema rkable e inter interindiv individual idual vari variabili abili- ty.27,60,62,63 However, by 6 or 12 months of age age,, var variat iation ion bet betwee ween n ind indivi ividua duals ls is

yet the res result ultss are con consis sisten tentt wit with h  the notion that early colonization by C per perfri fringen ngens  s   predisp predispose osess to lat later er deve de velo lopm pmen entt of NE NEC. C. Wa Wang ng et al66 completed the largest of the 4 cultureinde in depe pende ndent nt st stud udie iess of th the e mi micr croobiology of human NEC. In their study, 16S 16 S ge gene ne se sequ quen ence ce an anal alys ysis is wa wass used use d to cha charac racter terize ize the fec fecal al flor flora a of 10 pre preter term m inf infant antss wit with h NEC and 10 match matched ed contr controls. ols. These autho authors rs used us ed re rece cent ntly ly dev devel elop oped ed bi bioi oinf nfor or-matic techniques in conjunction with

stu study dyon ofthese gut microbe micr obess is nee needed ded to build groundbreaking preliminary studies to definitively determine if onset of NEC is associated with alterations in the structure of gut bacterial communities.

far less prono pronounced. unced. What these results reinforce is that the gut microbial communities of newborns are tenuouss and uns uou unstab table le rel relati ative ve to old older er children and adults. It is logical, therefore, to pursue the still unproven hypothes pot hesis is tha thatt cha change ngess wit within hin the these se fragile microbial communities correlate la te wi with th th the e ons nset et of mi miccro robe be-mediated diseases such as NEC. Thus far, 4 small studies have charac terized 16S ribosomal RNA gene sequences from gut bacteria of human

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 adminis tration of antibiotics or, perhaps,

The dramatic new opportunities now available to study intestinal microbes in th the e co cont ntex extt of NE NEC C ha have ve la larg rgel elyy result res ulted ed fro from m 2 dev develo elopme pments nts ove overr  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  extraord extr aordinary inary highhigh-throu throughput ghput technologies nolog ies with clea clearr relev relevance ance to en teric microbiology. microbiology. Recently developed sequ se quenc encin ing g pl plat atfo form rmss (e (eg, g, GS FL FLX X [Roche, Indianapolis, IN] and ABI SOLiD [Applied [Appl ied Biosy Biosystems stems Inc, Foste Fosterr City, CA]) CA ]) ar are e ma maki king ng it po poss ssib ible le to se se-quence que nce mas massiv sive e amo amount untss of DNA or

infantss wit infant with h NE NEC. C. Mil Millar lar et al64 published the first such study in 1996 and did not identify significant differences

secondary to the mucosal inflamma tion inherent to the primary disease process.

RNA iso isolat lated ed fro from m mic microb robial ial spe specicimens67; similarly important advances in mass spectrometry spectrometry have enabl enabled ed

780   MOROWITZ et al

APPLICATION OF HIGHTHROUGHPUT TECHNOLOGIES TO STUDY INTESTINAL MICROBES

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by all viruses that can be used for easy culture-i cultu re-independ ndependent ent speci species es ident identifiifi75 cation. However, if all viral DNAs and RNAss wit RNA within hin a bio biolog logica icall sam sample ple are sequenced, then the diversity and complexity of the viral community can be characterized accurately. Such an approach has been used in limited in-

FIGURE 1 DNA-based investigations of microbial community structure.

characterizations of thousands of microbial proteins or metabolic byproducts within biological specimens.68 Al though it was previously unfeasible to screen all or nearly all DNA, RNA, or protei pro teins ns wit within hin bio biolog logica icall sam sample pless from multiple patients, such investiga tions are now feasible and, in fact, underway. Metagenomics

crobiall pop crobia popula ulatio tion, n, and seq sequen uencin cing g metagenomes was not technically feasible before the recent advances in sequencing technolo technology. gy.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 mi-

stances to demonstrate that a large number of viruses within the gastroin testinal tract are actually bacteriophages pha ges tha thatt inf infect ect gut bac bacter teria. ia.76 A rece re cent nt st stud udyy de demo mons nstr trat ated ed th that at,, whereas meconium from a newborn infant did not contain viral particles, samples obtained 1 week later from  the same infant contained over 108 virall pa ra part rtic icle less per gr gram am of fe fece ces. s.77 Anothe Ano therr st study udy use used d a met metage agenom nomic ic approa app roach ch to ide identi ntify fy nov novel el vir viruse usess present within stool samples from pe78

Accumulating experience with the molecular lecul ar char character acterizati ization on of intes intestinal tinal microbes micr obes has clear clearly ly demon demonstra strated ted  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% 25%..70 For exa exampl mple, e, a 16S 16S-ba -based sed stud st udyy ma mayy id iden enti tify fy th the e pr pres esen ence ce of an E 

crobes ar crobes are e ess essent ential ial for deg degrad rading ing specifi spe cificc com compon ponent entss of the hum human an diet di et.. In ad addi diti tion on,, me meta tage geno nomi micc se se-quencin quen cing g has ide identi ntified fied the imp imporor tance of DNA sequences related to microbial crobi al virul virulence ence and hori horizonta zontall gene  transfer,73,74 both of which could be important to NEC pathogenesis. Successfully characterizing all microbial DNA within biological samples is substan tially more difficult when samples are complex. Because the neonatal intestinal tract contains far fewer speci species es

DNA-based inves DNA-based investigat tigations ions can delineate the genetic potential of a gut microbial community. However, it is well recognized that the presence of a par ticular microbial gene within the gut microb mic robiom iome e by no mea means ns ind indica icates tes  that the gene in question will be ex-

coli  species  species but would not provide any informati infor mation on about wheth whether er that part particicularr str ula strain ain of E co coli  li is pre predic dicted ted (on the basis of DNA content) to be a harmless commensal comm ensal or harm harmful ful patho pathogen. gen. 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 commun com munity ity fun functi ction on tha thatt are mad made e possible by sequencing the entire in testinal “metagenome.”

 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-throug high-t hroughput hput sequen sequencing cing conti continnues to decrease, it should become feasible to conduct an adequately powered ere d com compar pariso ison n of met metage agenom nomes es from infants with and without NEC.

pressed. press ed. For exam example, ple, docum documentin enting g  the presence of a gene encoding a pro tein involved in virulence or antibiotic resist res istanc ance e ind indica icates tes tha thatt the gen gene e might be expressed without indicating whethe whe therr the gen gene e act actual ually ly is exp expres ressed sed in a given biological sample. As such,  there is great interest in developing  technologies to assess the phenotype of microbial communities.

Metagenomics offers particular promise for investigations of viruses within  the gastrointesti gastrointestinal nal tract. In contrast

Three general lines of inquiry are now being applied to evaluate the function of naturally occurring microbial com-

The metagenome (or “community genome”) is defined as the combined genome no mess of al alll sp spec ecie iess wi with thin in a mi mixe xed d mi mi--

 to marker genes such as 16S that are present prese nt in all bacterial bacterial speci species, es, there are no RNA or DNA sequences shared

munities such as those found in the gastrointestinal tract (Fig 2). Each of   these approaches is routinely used

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diatric patie diatric patients nts with diarr diarrhea. hea. The clinical relevance of these findings is not yet known. Functional Characterizations of  Intestinal Microbial Communities

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FIGURE 2 High-throughput investigations of microbial community function.

between the gut microbial communi ties 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 group groups. s. 27,63,90 However, high throu ghputt molecular investigations  throughpu of this subject are only beginning to emerge and are likely to identify such differences.

(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 mo re di diffi fficu cult lt wh when en ap appl plyi ying ng su such ch  technologies to study naturally occurring biological biological samp samples les that contain unknown and often complex mixtures of micr microbial obial speci species. es. Metat Metatransc ranscriprip tomics, or “community transcriptom-

 the end products of gene and protein expression.86,87 Ea Each ch of th thes ese e hi high gh- throughput 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 infor informati mative ve with regard reg ard to mic microb robial ial func functio tion n els elseewhere in the gastrointestinal tract. Al though these approaches have not yet

ics,” 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 ge ne ex expr pres essi sion on in sa samp mple less fr from om ocean water and soil79–81; however, no published study has thus far detailed community gene expression within the intestinal intes tinal trac tract. t. Meta Metaprote proteomic omics, s, or “community proteomics,” is similarly  the study of proteins collectively expressed press ed withi within n micr microbial obial comm communiuni ties. Particularly when corresponding metagenomic data sets are available, mass-spectrometry– mass-spectromet ry– based proteomic analysis analy sis has enable enabled d the gener generation ation and subsequent validation of fascinating hypotheses regarding function of  previously previ ously unreco unrecognized gnized prot proteins. eins.82,83 Recently, 2 publications indicated that  this approach is feasible in the study of  gut mic microb robes es in hum humans ans..84,85 Finally, metabolomics is the study of low molecular weight microbial metabolites within withi n biolo biological gical samp samples. les. Identi Identificafica tion of metabolites within a gut micro-

been exploited to study the microbiology of NEC NEC,, the sim simpli plicit cityy of the juv juveni enile le intestinal tract may allow for successful completion of such studies in coming years.

cade ha cade hass be been en th that at nut nutri riti tion on,, gu gutt microbial function, and energy metabolism are tight tightly ly inter intertwine twined. d.4,88 This hass pa ha part rtic icul ular ar re rele leva vanc nce e to VL VLBW BW infa in fant nts, s, be beca caus use e th the e in inci cide denc nce e of  microb mic robe-m e-medi ediate ated d dis diseas ease e suc such h as NEC and lat late-o e-onse nsett sep sepsis sis is sig signifi nifi-cantlyy higher in form cantl formula-f ula-fed ed infan infants. ts.89 A critical and unresolved issue is how gut microbial communities differ in infants fan ts who rec receiv eive e art artific ificial ial for formul mula a and those who receive maternal milk. Because different dietary sources can

Multiple clinical studies have been performed to determine if the incidence of  NEC can be reduced by the administra tion  tio n of pro probiot biotics ics (defi (defined ned as die dietar taryy supplem supp lements ents of liv live e mic microo roorga rganism nismss  thatt are deriv  tha derived ed from from the human human intesti intesti-nal 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 

bial community arguably serves as the most accurate measure of microbial function by unambiguously measuring

be viewed as distinct “growth media” for int intest estina inall mic microb robes, es, it mak makes es sen sense se intuitively that differences must exist

and   Lactobacillus   is lo lowe werr wi withi thin n th the e gastrointest gastr ointestinal inal tracts of hospita hospitalized lized pret pr eter erm m in infa fant ntss th than an in ot othe herw rwis ise e

782   MOROWITZ et al

UNRESOLVED QUESTIONS RELATED TO INTESTINAL MICROBES AND NEC PATHOGENESIS Diet and the Intestinal Microbial Community

An imp import ortant ant les lesson son fro from m the pat pathhbreaking research over the past de-

We recently completed a metabolomic analysis of fecal samples from milkfed and formula-fed premature infants by using a mass-spectrometry– mass-spectrometry– based platform (V.P. and M.J.M., unpublished data da ta). ). We id iden enti tifie fied d a to tota tall of 17 174 4 ch chem em-ical compounds within these samples, and an d th the e di dist stri ribu buti tion on of ch chem emic ical alss within these samples was sharply affected by die fected diet. t. Pri Princi ncipal pal com compon ponent ent analysis of the data sets demonstrated  thatt sam  tha samples ples fro from m mil milk-fe k-fed d and for formula mula-fed infa infants nts clu cluste stered red tig tightly htly tog togethe ether. r. These Thes e res results ults indicate indicate that the act active ive metabolic pathways used by microbes within wit hin the int intest estinal inal tra tracts cts of mil milk-fe k-fed d and formula-fed infants are distinct. The applica appl icatio tion n of othe otherr high high-th -throu roughpu ghputt  technol  tec hnologi ogies es will help help further further define define the impact of diet on the gut microbial communities of newborn infants. Probiotics and the Intestinal Microbial Community

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CONCLUSIONS

healthy term infants infants..15 A recent metaanalysis concluded that enteral supplementation of probiotics does reduce the risk ri sk of dev develo elopi ping ng NE NEC C fo forr lo low w bir birth th 91 weight weig ht infa infants. nts. Alt Althoug hough h nume numerou rouss questions remain regarding safety, dosage, and the specific species used in different formulations, the prospect of a

(which may be possible with studies now no w in pr prog ogre ress ss)) wo woul uld d gr grea eatl tlyy strength stre ngthen en the rati rational onale e for usin using g probiotics. However, if the pathogenesis of NEC is truly related to a deficiency of favorable bacteria, and if   this defi deficien ciency cy is share shared d by all premature mat ure inf infant ants, s, one mus mustt won wonder der

new strategy to prevent onset of NEC is exciting. Nonetheless, Nonethel ess, ther there e are seve several ral important points to be made about probiot bi otic icss an and d NE NEC C in th the e co cont ntex extt of  emerging technologies to characterize int intest estina inall mi micr crobe obes. s. Fir First st,, the purported deficiency of “beneficial” gut bacteria in premature infants is based on older, culture-based studies and, thus, should be viewed with cauti ca ution. on. Ong Ongoi oing ng st studi udies es to doc docuument colonization patterns with mo-

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 pati patients ents at risk. The diffi difficult culty, y, of course, lies in prospectively iden tifying  tifyi ng whic which h pati patients ents are at high highest est risk for developing the disease. At present, there is no effective method  to ascertain asce rtain which 5% to 10% 1 0% of VLBW VL BW infants will develop the disease. The-

Recent advances in the study of intes tinal microbes have demonstrated  that the microbial ecosystem within  the human gastrointestinal tract is exceedingly ceedi ngly complex. However, the advent ve nt of ne new w hi high gh-t -thr hrou ough ghpu putt te tech ch-nologi nol ogies es has led to pro promis mising ing new opportunit oppor tunities ies to study the contr contributi ibution on 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 con tribute to disease pathogenesis. The gut micr microbial obial ecosystems ecosystems of prema prema- ture infants are uniquely amenable to  the use of high-throughput technolo-

lecularr tec lecula techni hnique quess wi will ll be par parti ticcular ul arly ly va valu luab able le in co confi nfirm rmin ing g 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

oretically, 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 feasi feasible ble to give probiotics to a select group of pa tients  tien ts at high risk rathe ratherr than to all patients.

giess suc gie such h as met metage agenom nomics ics bec becaus ause e of   the lack of microbial diversity in the newborn intestine. Application of these  technol  tec hnologi ogies es to biolog biologica icall samples samples from from affected and unaffected newborns may lead to advances in the care of low birth weig we ight ht inf infant antss who ar are e at ri risk sk fo forr th the e unabated problem of NEC.

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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

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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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