Probiotics – PrebioticsNovel Strategies That May Prevent Neonatal Disease

Richard J. Schanler, M.D.Schneider Children’s Hospital at North Shore, Manhasset, NY

and Albert Einstein College of Medicine, Bronx, NYschanler@nshs.edu

February 2010

Intestinal EcosystemIntestinal Ecosystem Host cellsHost cells NutrientsNutrients MicrofloraMicroflora

Interactions with nutrients and cellsInteractions with nutrients and cells Adult colon and distal small intestineAdult colon and distal small intestine

10101313 microorganisms, 500 species microorganisms, 500 species Normal bacterial flora affect immune functionNormal bacterial flora affect immune function

Stimulate mucosal IgA productionStimulate mucosal IgA production Influence gene expressionInfluence gene expression Activate toll-like receptors to protect against gut Activate toll-like receptors to protect against gut

injuryinjury

10 trillion10 trillion

Newborn Intestinal Flora Colonization in 12 to 24 hours

Enterobacteria, E. coli Enterococci Anaerobes

Formula-fed infants Same as above Bifidobacteria

Breastfeeding infants Bifidobacteria and

Lactobacilli compete with above

Premature infants delayed colonization of commensal microflora

Antibiotics Delayed feeding Sterile environment

Some of the Intestinal Microflora Some of the Intestinal Microflora Identified in Infants are BeneficialIdentified in Infants are Beneficial

BifidobacteriuBifidobacteriumm

LactobacillusLactobacillus StaphylococcusStaphylococcus StreptococcusStreptococcus BacteroidesBacteroides ClostridiumClostridium EscherichiaEscherichia EnterococcusEnterococcus VellonellaVellonella PropionibacteriuPropionibacteriu

mm BacillusBacillus EubacteriumEubacterium

B. adolescentisB. adolescentis B. bifidumB. bifidum B. breveB. breve B. catenulatumB. catenulatum B. globosumB. globosum B. infantisB. infantis B. longumB. longum B. suisB. suis

L. caseiL. casei L. salivariusL. salivarius L. gasseriL. gasseri L. planarumL. planarum L. fermentumL. fermentum

Functions of Commensal MicroorganismsFunctions of Commensal Microorganisms Probiotic speciesProbiotic species Infection protectionInfection protection

Competition with pathogensCompetition with pathogens Substrates, receptorsSubstrates, receptors

BacteriocinsBacteriocins MetabolismMetabolism

Fermentation, SCFA, vitaminsFermentation, SCFA, vitamins Conversion of procarcinogensConversion of procarcinogens

Immune responseImmune response IgA synthesisIgA synthesis Interaction with intestinal immune systemInteraction with intestinal immune system

Growth facilitated by oligosaccharidesGrowth facilitated by oligosaccharides Human milkHuman milk ““Prebiotics”Prebiotics”

Intestinal HomeostasisIntestinal Homeostasis

Jesse N, Neu J 2006; Lin 2008Commensal bacteria reduce inflammatory signaling in intestinal epithelium via inhibition of NF-B pathway.

Carbohydrate FermentationCarbohydrate Fermentation Major function of intestinal bacteriaMajor function of intestinal bacteria Bifidobacteria and lactobacilli Bifidobacteria and lactobacilli

ferment carbohydrate to produce lactic ferment carbohydrate to produce lactic acidacid

Acidic milieu potentiates nonpathogenic Acidic milieu potentiates nonpathogenic bacteriabacteria

Coliforms Coliforms ferment carbohydrate to COferment carbohydrate to CO22 and water and water neutral pHneutral pH

Stool pH: Indicator of Normal Gut

Fermentation

4.8

5

5.2

5.4

5.6

5.8

6

3rd Day 1stMonth

2ndMonth

3rdMonth

4thMonth

BreastfedFormula-Fed

Indrio et al. Pediatr Res 2007; 62:98

Approaches to Create Intestinal Approaches to Create Intestinal Microflora Like Breastfed InfantMicroflora Like Breastfed Infant

Two possible approaches:Two possible approaches:1.1. ProbioticsProbiotics

Feed live microorganismsFeed live microorganisms2.2. PrebioticsPrebiotics

Provide food for the beneficial bacteriaProvide food for the beneficial bacteria

FAO/WHO Working Group: Guidelines for the Evaluation of Probiotics in Food. 2002:

URL:http://www.who.int/foodsafety/publications/fs_management/probiotics2/en/index.html.

ProbioticsProbiotics

ProbioticsProbiotics 1900s Elie Metchnikoff, Pasteur 1900s Elie Metchnikoff, Pasteur

InstituteInstitute ““Long life span of Bulgarian Long life span of Bulgarian

peasants due to ingestion of peasants due to ingestion of fermented milks containing large fermented milks containing large numbers of nonpathogenic numbers of nonpathogenic bacteria”bacteria”

ProbioticsProbiotics Antonym to the term ‘antibiotics’Antonym to the term ‘antibiotics’ Live, nonpathogenic bacterial micro-organismsLive, nonpathogenic bacterial micro-organisms Colonize host intestineColonize host intestine Bolster natural host defensesBolster natural host defenses TypesTypes

LactobacillusLactobacillus BifidobacteriumBifidobacterium SaccharomycesSaccharomyces Streptococcus thermophilusStreptococcus thermophilus

Characteristics of an Ideal ProbioticCharacteristics of an Ideal Probiotic Stable to acids & bile saltsStable to acids & bile salts Adhesive to intestinal epithelial cellsAdhesive to intestinal epithelial cells NoninvasiveNoninvasive NonpathogenicNonpathogenic Immuno-stimulatoryImmuno-stimulatory Not pro-inflammatoryNot pro-inflammatory Antagonistic to pathogensAntagonistic to pathogens Sensitive to usual antibioticsSensitive to usual antibiotics Doesn’t develop resistanceDoesn’t develop resistance Originate from human microfloraOriginate from human microflora

Probiotics Enhance Host DefenseProbiotics Enhance Host Defense Prevent colonization by pathogenic Prevent colonization by pathogenic

organismsorganisms Competition for nutrientsCompetition for nutrients Competition for attachment sitesCompetition for attachment sites

Produce antibacterial compounds Produce antibacterial compounds unfavorable to pathogensunfavorable to pathogens Volatile fatty acidsVolatile fatty acids Modified bile acidsModified bile acids

Recruit immune cellsRecruit immune cells Activate immune and inflammatory Activate immune and inflammatory

responsesresponses

Probiotics Enhance Gut Barrier FunctionProbiotics Enhance Gut Barrier Function Intestine is largest immune organ in the bodyIntestine is largest immune organ in the body Large surface barrier Large surface barrier Gut permeabilityGut permeability TranslocationTranslocation

Passage of intestinal bacteria through intestinal Passage of intestinal bacteria through intestinal mucosamucosa

To extra-intestinal sitesTo extra-intestinal sites Intestinal mucosal barrierIntestinal mucosal barrier

Physical, cellular barrierPhysical, cellular barrier Major role in development of NECMajor role in development of NEC

Lactic acid bacteria [Probiotic]Lactic acid bacteria [Probiotic] Do not translocateDo not translocate Prevent translocation of other bacteriaPrevent translocation of other bacteria Produce antimicrobial bacteriocinsProduce antimicrobial bacteriocins

Probiotics Improve Enteral NutritionProbiotics Improve Enteral Nutrition Improve mucosal integrityImprove mucosal integrity

Increase mucous secretionIncrease mucous secretion Enhance GI motilityEnhance GI motility Aid in intestinal maturationAid in intestinal maturation

Improve feeding toleranceImprove feeding tolerance Metabolize noxious substancesMetabolize noxious substances Produce protective nutrientsProduce protective nutrients

Arginine, glutamine, short chain fatty acidsArginine, glutamine, short chain fatty acids Synthesize vitamin KSynthesize vitamin K

How do Probiotics Work?How do Probiotics Work? Competitive exclusionCompetitive exclusion

Direct effectDirect effect bacteriocidal factorsbacteriocidal factors mucin productionmucin production pHpH

Epithelial barrierEpithelial barrier bacterial translocationbacterial translocation antigen translocationantigen translocation

Anti-inflammatoryAnti-inflammatory IL-10 IL-10 ubiquinationubiquination IL-8IL-8 tumor necrosis factor (TNF)tumor necrosis factor (TNF)

InflammationInflammation IgAIgA IL-6IL-6 NK call activityNK call activity phagocytosisphagocytosis -defensin-2-defensin-2

Innate immunityInnate immunity Mannose receptorsMannose receptors Toll-like receptorsToll-like receptors

TR-2: gm+TR-2: gm+ TR-4: gm-TR-4: gm-

Probiotics and Antibiotic-Associated Probiotics and Antibiotic-Associated DiarrheaDiarrhea

Children 6 mo – 10 yChildren 6 mo – 10 y US midwestUS midwest Oral antibiotics x 10 dOral antibiotics x 10 d RandomizationRandomization Supplement x1/daySupplement x1/day

Lactobacillus GG, 1-2 Lactobacillus GG, 1-2 x10x1099

PlaceboPlacebo Diarrhea incidenceDiarrhea incidence

Lactobacillus: 7 cases Lactobacillus: 7 cases (8%)(8%)

Placebo: 25 cases (26%)Placebo: 25 cases (26%) Diarrhea durationDiarrhea duration

Lactobacillus: 4.7 daysLactobacillus: 4.7 days Placebo: 5.9 daysPlacebo: 5.9 days

Vanderhoof, J Pediatr 1999; 135:564

Stool Consistency Log

Probiotics Reduce Diarrhea in Probiotics Reduce Diarrhea in Children: Five Meta-AnalysesChildren: Five Meta-Analyses

No. trialsNo. trials No. No. subjectssubjects

OutcomeOutcome

#1:#1: 2323 1,9001,900 risk diarrhearisk diarrhea>3 days>3 days

#2#2 99 765765 duration: 0.7 dduration: 0.7 d

#3#3 1313 1,1131,113 duration: 0.8 dduration: 0.8 d

#4#4 55 619619 duration: 1.1 dduration: 1.1 d

#5#5 88 988988 duration: 1.1 d duration: 1.1 d 1H. Szajewska & J. Mrukowicz JPGN 2001;33:S17-S252C. Van Niel et al. Pediatrics 2002;109:S678-6843J. Huang et al. Dig Dis Sci 2002;1:2626-26344H. Szajewska et al. APT 2007;25:257-2645H. Szajewska et al. APT 2007;25:871-881

Necrotizing EnterocolitisNecrotizing Enterocolitis GI catastropheGI catastrophe PrematurityPrematurity Bacterial floraBacterial flora

Reduction in commensal bacteria Reduction in commensal bacteria More pathogenic than nonpathogenicMore pathogenic than nonpathogenic

Immature intestineImmature intestine Impaired mucosal barrier functionImpaired mucosal barrier function Increased GI permeabilityIncreased GI permeability

Rationale for Probiotics in NECRationale for Probiotics in NEC Lactobacilli and Bifidobacteria have low rates of Lactobacilli and Bifidobacteria have low rates of

spontaneous colonization in breastfed infants spontaneous colonization in breastfed infants <1500 g<1500 g

Probiotics reduce pathogenic bacterial Probiotics reduce pathogenic bacterial colonizationcolonization Multiple courses of antibioticsMultiple courses of antibiotics Abnormal NICU floraAbnormal NICU flora Microbial etiologies in NEC Microbial etiologies in NEC

Reinforce intestinal barrierReinforce intestinal barrier Prevent bacterial translocationPrevent bacterial translocation

Alleviate intestinal inflammationAlleviate intestinal inflammation Balance of anti- and pro-inflammatory Balance of anti- and pro-inflammatory

cytokinescytokines

Probiotics and NEC - Animal Probiotics and NEC - Animal StudiesStudies

Bifidobacteria vs control Bifidobacteria vs control Rat modelRat model

Gut colonization by 24 hoursGut colonization by 24 hours Asphyxia/hypothermia model for NECAsphyxia/hypothermia model for NEC Reduction in NEC with probiotics Reduction in NEC with probiotics

Bifidobacteria 30%Bifidobacteria 30% Control 70%Control 70%

Probiotics and Premies – JapanProbiotics and Premies – Japan Randomized, controlled trial, JapanRandomized, controlled trial, Japan VLBW infants, N=91VLBW infants, N=91 Bifidobacterium breve Bifidobacterium breve supplement vs controlsupplement vs control Supplement groupSupplement group

Higher fecal colonization of bifidobacteria at 2 wkHigher fecal colonization of bifidobacteria at 2 wk 73 % vs 12%73 % vs 12%

Tolerated more milk feedingTolerated more milk feeding Fewer gastric residualsFewer gastric residuals Improved overall feeding toleranceImproved overall feeding tolerance Improved weight gainImproved weight gain

Kitajima, Arch Dis Child 1997;76:F101

Probiotic year, n=1237 infantsProbiotic year, n=1237 infants Infloran:Infloran:

2.5 x 102.5 x 1088 Lactobacillus acidophilusLactobacillus acidophilus 2.5 x 102.5 x 1088 Bifidobacteria infantisBifidobacteria infantis

Historical controls, n=1282 infantsHistorical controls, n=1282 infants Prior year data on morbidityPrior year data on morbidity

Probiotics and Premies - BogotaProbiotics and Premies - Bogota

Hoyos, Int J Inf Dis 1999; 3:197Hoyos, Int J Inf Dis 1999; 3:197

ResultsResults NEC incidenceNEC incidence

Probiotic year: 37/1237 Probiotic year: 37/1237 3% 3% Historical control year: 85/1282 Historical control year: 85/1282 6.6% 6.6%

NEC mortality – significant differenceNEC mortality – significant difference Probiotic year Probiotic year 14 cases 14 cases Historical control year Historical control year 35 cases 35 cases

Probiotics and Premies - BogotaProbiotics and Premies - Bogota

Hoyos, Int J Inf Dis 1999; 3:197Hoyos, Int J Inf Dis 1999; 3:197

Probiotics and Premies - ItalyProbiotics and Premies - Italy Double blind, 12 centers in ItalyDouble blind, 12 centers in Italy Infants < 33 wk or < 1500 grams; n=585Infants < 33 wk or < 1500 grams; n=585

Mean BW 1335 g, GA 31 wks; 64% rec’d human Mean BW 1335 g, GA 31 wks; 64% rec’d human milkmilk

Lactobacillus GGLactobacillus GG supplement supplement (Dicoflor, Dicofarm, IT)(Dicoflor, Dicofarm, IT) vs vs controlcontrol Once daily (6 x 10Once daily (6 x 1099) from onset feeding to ) from onset feeding to

dischargedischarge Supplementation for ~50 daysSupplementation for ~50 days

Supplement ControlSupplement Control UTIUTI 3.4%3.4% 5.8%5.8% nsns NECNEC 1.4 %1.4 % 2.7%2.7% nsns SepsisSepsis 4.4 %4.4 % 3.8 %3.8 % nsns

Dani, Biol Neonate 2002; 82:103

Probiotics and Premies - IsraelProbiotics and Premies - Israel Randomized, double-blind studyRandomized, double-blind study Daily supplement, first feed to 36 wk Daily supplement, first feed to 36 wk

corrected agecorrected age ABC Dophilus (Solgar, Israel)ABC Dophilus (Solgar, Israel)

Bifidobacteria infantis Bifidobacteria infantis 0.35 x 100.35 x 1099 cfu/daycfu/day

Bifidobacteria bifidus Bifidobacteria bifidus 0.35 x 100.35 x 109 9 cfu/daycfu/day Strep thermophilus Strep thermophilus 0.35 x 100.35 x 109 9 cfu/daycfu/day

Power analysis Power analysis n=145 for reduction in NEC from 15% to n=145 for reduction in NEC from 15% to

5%5%Bin-Nun et al, J Pediatr 2005;147:192-6

Probiotics ControlProbiotics ControlProbiotics and Premies - IsraelProbiotics and Premies - Israel

Birth weight (g)Birth weight (g) 11521152 11111111Gestation (wk)Gestation (wk) 29.829.8 29.329.3SGA (%)SGA (%) 2525 1515Sepsis (%)Sepsis (%) 4343 3333 nsnsAntibiotics (d) Antibiotics (d) 12.512.5 14.914.9Full feeding (d)Full feeding (d) 14.614.6 17.517.5TPN discontinued (d)TPN discontinued (d) 16.616.6 18.618.6Human milk exclusive (%)Human milk exclusive (%) 5858 6464Partial human milk (%)Partial human milk (%) 1717 1212Formula (%)Formula (%) 2525 2323NPO (d)NPO (d) 5.25.2 4.84.8

All differences are not significant Bin-Nun et al, J Pediatr 2005;147:192-6

Probiotics, Premies, NEC - IsraelProbiotics, Premies, NEC - Israel Probiotics Control Probiotics Control P valueP value

††Bell stage of NEC 1-3 All differences are significant Bell stage of NEC 1-3 All differences are significant Bin-Nun et al, J Pediatr 2005;147:192-6Bin-Nun et al, J Pediatr 2005;147:192-6

NEC cases (n, %)NEC cases (n, %) 3 (4%)3 (4%) 12 (16%)12 (16%) 0.030.03

Stage at diagnosisStage at diagnosis†† 1.31.3 2.32.30.0050.005

NEC or death (n, %)NEC or death (n, %) 6 (8%)6 (8%) 17 (23%)17 (23%)0.0250.025

Probiotics, Premies, & NEC - Probiotics, Premies, & NEC - TaiwanTaiwan

Randomized, controlled, blinded trial, infants <1500 Randomized, controlled, blinded trial, infants <1500 gramsgrams Mean 1100 g, ~28 wksMean 1100 g, ~28 wks

Probiotic: InfloranProbiotic: InfloranTMTM with breastmilk twice daily, with breastmilk twice daily, n=180n=180 Lactobacillus acidophilusLactobacillus acidophilus Bifidobacteria infantisBifidobacteria infantis

Control group: breastmilk only, n=187Control group: breastmilk only, n=187 Breastmilk: either mother’s own or donor human Breastmilk: either mother’s own or donor human

milkmilkLin et al, Pediatrics 2005;115:1-4

Probiotics, Premies, & NEC - Probiotics, Premies, & NEC - TaiwanTaiwan

Probiotics Control Probiotics Control P P valuevalue

Death, n (%)Death, n (%) 7 (3.9)7 (3.9) 10 (10.7)10 (10.7).009.009

NEC or Death cases, n (%)NEC or Death cases, n (%) 9 (5)9 (5) 24 (12.8)24 (12.8).009.009

NEC, stage 2 or 3, n (%)NEC, stage 2 or 3, n (%) 2 (1.1)2 (1.1) 10 (5.3)10 (5.3).04.04

Sepsis, n (%)Sepsis, n (%) 22 (12.2)22 (12.2) 36 (19.3)36 (19.3).03.03

NEC or sepsis, n (%)NEC or sepsis, n (%) 24 (13.3)24 (13.3) 46 (24.6)46 (24.6).03.03

NEC, sepsis, or death, n (%)NEC, sepsis, or death, n (%) 31 (17.2)31 (17.2) 60 (32.1)60 (32.1).009.009

Lin et al, Pediatrics 2005;115:1-4

Probiotics, Premies, & NEC – Taiwan IIProbiotics, Premies, & NEC – Taiwan II Randomized, controlled, masked trial, infants 500-Randomized, controlled, masked trial, infants 500-

1500 g1500 g MulticenterMulticenter Mean birth weight 1100 g, n=217 per groupMean birth weight 1100 g, n=217 per group Breastmilk or mixed dietBreastmilk or mixed diet Intervention: InfloranIntervention: InfloranTMTM with milk twice daily x 6 wk with milk twice daily x 6 wk

Lactobacillus acidophilus Lactobacillus acidophilus 101099

Bifidobacteria infantis Bifidobacteria infantis 101099

Control groupControl group

Lin H-C et al, Pediatrics 2008;122:693-700

Probiotics, Premies, & NEC – Taiwan IIProbiotics, Premies, & NEC – Taiwan II

Probiotics Control Probiotics Control P P valuevalue

Birth weight (g)Birth weight (g) 1029 1029 ± 246± 246 1077 1077 ±± 214 214 .03.03

Death or NEC cases, n (%)Death or NEC cases, n (%) 4 (1.8)4 (1.8) 20 (9.2)20 (9.2).002.002

NEC, stage NEC, stage >> 2, n (%) 2, n (%) 4 (1.8)4 (1.8) 14 (6.5)14 (6.5) .02.02

Sepsis, n (%)Sepsis, n (%) 40 (19.8)40 (19.8) 24 (11.5)24 (11.5) .04.04

Gram Positive, n (%)Gram Positive, n (%) 25 (11.5)25 (11.5) 19 (8.8)19 (8.8) NSNS

Gram Negative, n (%)Gram Negative, n (%) 15 (6.9)15 (6.9) 5 (2.3)5 (2.3).037.037

Lin H-C et al, Pediatrics 2008;122:693-700

Adjusted Odd’s Ratio for NEC or Death: 5.6 (1.8 – 17.9)Adjusted for birth weight, surfactant, pH, gestational age, site

Adjusted Odd’s Ratio for Sepsis: 0.63 (0.35 - 1.10)Adjusted for birth weight, umbilical catheters, ventilation, NICU stay, site

Probiotics and Time to Full FeedingsProbiotics and Time to Full Feedings

WMD = weighted mean difference, probiotic - controlProbiotic therapy shortens time to full feeding, P = 0.02Deshpande G, Rao S, Patole S. Lancet 2007; 369:1614-1620

ProbioticsProbiotics Control Control WMD (95% WMD (95% CI)CI)

Kitajima 2007 Kitajima 2007 4545 17 17 ±± 8.48.4 464620.5 ± 11.920.5 ± 11.9 -3.9 (-7.7, 0.7)-3.9 (-7.7, 0.7)

Bin Nun 2005Bin Nun 2005 727214.6 14.6 ± 8.7± 8.7 7373 17.5 ± 13.617.5 ± 13.6-2.9 (-6.6, 0.8)-2.9 (-6.6, 0.8)

ManManzoni 2006zoni 2006 3939 15 15 ±± 8 8 4141 17 17 ±± 9 9-2 (-5.7, 1.7)-2 (-5.7, 1.7)

TOTALTOTAL 156156 160160-2.7 (-5, -0.5)-2.7 (-5, -0.5)

Probiotics, Premies, & NECProbiotics, Premies, & NEC BogotaBogota 6.6 6.6 3% 3%

Lactobacillus acidophilusLactobacillus acidophilus Bifidobacteria infantisBifidobacteria infantis

ItalyItaly 2.7 2.7 1.4% ns 1.4% ns Lactobacillus GGLactobacillus GG

TaiwanTaiwan 5.3 5.3 1.1% 1.1%6.5 6.5 1.8% 1.8%

Lactobacillus acidophilusLactobacillus acidophilus Bifidobacteria infantisBifidobacteria infantis

IsraelIsrael 16 16 4 % 4 % Bifidobacteria infantisBifidobacteria infantis Bifidobacteria bifidusBifidobacteria bifidus Streptococcus thermophilusStreptococcus thermophilus

Randomized trials

Meta-Analysis Probiotics & NECMeta-Analysis Probiotics & NECProbioticsProbiotics Control Control RR (95% RR (95%

CI)CI)NEC NEC >> Stage II Stage II 13/637 13/637 37/627 37/627

0.32 (0.17, 0.60)0.32 (0.17, 0.60)

All MortalityAll Mortality 16/60116/601 39/60639/6060.43 (0.25, 0.75)0.43 (0.25, 0.75)

NEC-Related Mortality*NEC-Related Mortality* 0/3670/367 5/3635/3630.17 (0.02, 1.37)0.17 (0.02, 1.37)

SepsisSepsis 61/64761/647 97/63797/6370.93 (0.73, 1.19) 0.93 (0.73, 1.19) No differences in hospital days, parenteral nutrition days, or weight gain

AlFaleh KM, Bassler D. Cochrane Database of Systematic Reviews 2008*p = 0.03 by Fisher

Probiotics & MortalityProbiotics & Mortality

RR = relative riskProbiotic group had lower overall mortality, p = 0.0007Deshpande G, Rao S, Patole S. Lancet 2007; 369:1614-1620

ProbioticsProbiotics Control Control RR (95% RR (95% CI)CI)Kitajima 1997 Kitajima 1997 0/45 0/45 2/46 2/46

0.20 (0.01, 4.14)0.20 (0.01, 4.14)Dani 2002Dani 2002 12/29512/295 22/29022/290

0.54 (0.27, 1.06)0.54 (0.27, 1.06)Bin Nun 2005Bin Nun 2005 3/723/72 9/739/73

0.34 (0.10, 1.20)0.34 (0.10, 1.20)Lin 2005Lin 2005 7/1807/180 20/18720/187

0.36 (0.16, 0.84)0.36 (0.16, 0.84)Manzoni 2006Manzoni 2006 5/395/39 6/416/41

0.88 (0.29, 2.64) 0.88 (0.29, 2.64)

TOTALTOTAL 27/63127/631 59/63759/6370.47 (0.30, 0.73)0.47 (0.30, 0.73)

Lin 2008Lin 2008 2/2172/217 9/2179/217

Proposed Strategies for Preventing Proposed Strategies for Preventing NECNEC

Human milk feedingHuman milk feeding 1515 Enteral antibioticsEnteral antibiotics 1111 Fluid restriction [judicious]Fluid restriction [judicious] 1212 Enteral IgG and IgAEnteral IgG and IgA 1515 Antenatal steroidsAntenatal steroids 5454 Delayed or slow feedingDelayed or slow feeding not efficaciousnot efficacious Enteral IgG onlyEnteral IgG only not efficaciousnot efficacious Probiotics Probiotics

Lactobacillus GGLactobacillus GG 7777 Infloran (2 organisms)Infloran (2 organisms) 2424 Infloran (2 organisms)Infloran (2 organisms) 2121 Infloran (2 organisms)Infloran (2 organisms) 2020 ACDophilus (3 organisms)ACDophilus (3 organisms) 88

StrategyStrategy Number Needed To Treat Number Needed To Treat

Adapted from Bell, Pediatrics 2005;115:173

24

Probiotics: Unanswered IssuesProbiotics: Unanswered Issues TypeType

Which organism or combination of organisms?Which organism or combination of organisms? DosageDosage Duration of therapyDuration of therapy Frequency of dosingFrequency of dosing SafetySafety

Short-termShort-term Long-termLong-term

Mechanism of actionMechanism of action Combination with human milkCombination with human milk Combinations with prebioticsCombinations with prebiotics

Potential ComplicationsPotential Complications InfectionInfection

SepsisSepsis MeningitisMeningitis Soft tissueSoft tissue EndocarditisEndocarditis

Dental cariesDental caries Acquired bacterial resistanceAcquired bacterial resistance

LactobacillusLactobacillus Sepsis Sepsis Compromised infant hosts Compromised infant hosts Multiple courses of antibiotics Multiple courses of antibiotics A. Developed secondary diarrhea, treated with A. Developed secondary diarrhea, treated with Lactobacillus GGLactobacillus GG

Within 3 weeks of continued treatment Within 3 weeks of continued treatment patients developed fever patients developed fever systemic symptoms of sepsissystemic symptoms of sepsis gram positive anaerobic non-spore forming rods gram positive anaerobic non-spore forming rods isolated from bloodisolated from blood sensitive to penicillin [gentamicin for synergy]sensitive to penicillin [gentamicin for synergy]

B. Other cases NOT associated with probiotic therapyB. Other cases NOT associated with probiotic therapy

Land et al, Pediatrics 2005;115:178Thompson, J Perinatol 201;21:258

Boyle, et al. AJCN, 2005

What to do before multicenter trial Dose response in animals

Evaluate inflammatory pathways in animals

Evaluate comparative efficacies in animals

Evaluate live, heat killed and bacterial products for safety and efficacy

PrebioticsPrebiotics

PrebioticsPrebiotics Nondigestible, food ingredientsNondigestible, food ingredients Not absorbedNot absorbed Stimulate growth of beneficial fecal microflora, to improve healthStimulate growth of beneficial fecal microflora, to improve health Act as substrates for probiotics Act as substrates for probiotics ExamplesExamples

Oligosaccharides (from human milk)Oligosaccharides (from human milk) Act to increase bifidobacteria (bifidogenic effect)Act to increase bifidobacteria (bifidogenic effect) Inhibit intestinal binding of pathogenic bacteriaInhibit intestinal binding of pathogenic bacteria

Receptor analogues for glycoconjugates on intestinal surfacesReceptor analogues for glycoconjugates on intestinal surfaces Mediate cell to cell interactionMediate cell to cell interaction Bind humoral mediatorsBind humoral mediators Modulate signal transferModulate signal transfer Bind viruses and bacteriaBind viruses and bacteria

Kunz 1999, Newburg 2000, Coppa 1997, Brand-Muller 1998

Oligosaccharides: 3rd Most Common Component in Mature Human Milk

010203040506070

Lactose

FatOligosaccharides

Protein

Upper Range of ConcentrationLower Range of Concentration

g/L

Adapted from Kunz et al, Clin Perinatol 1999;26:307

Human Milk OligosaccharidesHuman Milk Oligosaccharides Colostrum – highest concentrationColostrum – highest concentration Mature milk – lower contentMature milk – lower content Contain galactose, glucose, N-acetylglucosamine, Contain galactose, glucose, N-acetylglucosamine,

fucose, sialic acidfucose, sialic acid Highly variable molecular structureHighly variable molecular structure Genetic variationGenetic variation

A,B,AB blood typesA,B,AB blood types Lewis blood groupLewis blood group

Human Milk OligosaccharidesHuman Milk Oligosaccharides Receptor analogues for glycoconjugates on Receptor analogues for glycoconjugates on

epithelial and endothelial cell surfacesepithelial and endothelial cell surfaces Mediate cell to cell interactionMediate cell to cell interaction Bind humoral mediatorsBind humoral mediators Modulate signal transferModulate signal transfer Bind viruses and bacteria Bind viruses and bacteria

HMO found in infant stoolsHMO found in infant stools Fermentation productsFermentation products

Kunz 1999, Newburg 2000, Coppa 1997, Brand-Muller 1998

Currently Available Prebiotics Currently Available Prebiotics Oligosaccharides (Not of Human Milk origin)Oligosaccharides (Not of Human Milk origin)

LactuloseLactulose Galacto-oligosaccharides (GOS)Galacto-oligosaccharides (GOS) Fructo-oligosaccharides (FOS)Fructo-oligosaccharides (FOS) InulinInulin Isomalto-oligosaccharidesIsomalto-oligosaccharides Soybean oligosaccharidesSoybean oligosaccharides LactosucroseLactosucrose Gluco-oligosaccharidesGluco-oligosaccharides Xylo-oligosaccharidesXylo-oligosaccharides

Oligosaccharides GOS and FOS differ from HMO. HMO contain lactose at their reducing end. They can be elongated with n = 0-15 lactosamine units and can be modified with 1 or more fucose residues in various linkages and/or carry N-acetylneuraminic acid in various linkages. Galactooligosaccharides (GOS) and Fructooligosaccharides (FOS) are polymers of galactose and fructose, respectively. Fructose is not naturally found in human milk. N-acetylneuraminic acid and fucose, which are abundant constituents of HMO and maybe be important for some HMO functions, are not part of GOS or FOS.

OligosaccharidesOligosaccharides

Stool Microflora After 6 wks Feeding Formula with/without Oligosaccharides

BIF EC CLOS ENT

%

0

10

20

30

40

50

60

70Formula without Oligosaccharides

Knoll, 2003; microflora by FISH analyses; Oligo supplement was FOS and GOSBIF=Bifidobacteria, EC=E. coli, CLOS=Clostridium, ENT=Enterobacteria**All differences significant, p < 0.05

Formula with Oligosaccharides

* *

* *

Effect of Prebiotics on Stool pHBreastfed Prebiotic Control Formula

5.9

6

6.1

6.2

6.3

6.4

6.5

pH

Scholtens, J Nutr 2008; 138:1141 analyses at 26 wks of studyDifferences in pH: Prebiotic vs Control, p < 0.05

Prebiotics Increase Fecal IgA Breastfed Prebiotic Control Formula

g/g feces

0

1000

Scholtens, J Nutr 2008; 138:1141 significant differences at 26 wks of study*Prebiotic vs Control, p < 0.05

*

Prebiotics Affect Infant GI TractPrebiotics Affect Infant GI Tract 271 infants studied271 infants studied 4 treatments4 treatments

BreastfedBreastfed Standard formulaStandard formula Formula with GOS (2.4 g/L)Formula with GOS (2.4 g/L) Breastfed and added GOS (2.4 g/L)Breastfed and added GOS (2.4 g/L)

Stool characteristicsStool characteristics Softer, more frequent stoolsSofter, more frequent stools

ToleranceTolerance Crying, spit-up, vomiting not affectedCrying, spit-up, vomiting not affected

Ben XM et al. Chin Med J (English). 2004;117(6):927-931

GOS/FOS (9:1) Decreased Episodes of Infection and Fever in Infants

Prebiotic Formula (GOS:FOS, 9:1), n=66

Control Formula, n=68

*

0

1

2

3

4

5

6

OveralInfections

URTI Infectionstreated withantibiotics

Feverepisodes

* *

*p < 0.01Number of

Episodes per Infant

High risk atopic disease. Hydrolysate formula with GOS&FOS 8 g/L, Intervention Birth to 6 months; follow-up to 2 years Arslanoglu S, et al. J Nutr 2008;138:1091-1095

GOS/FOS (9:1) Decreased Incidence of Allergic Manifestations

*

*

*

Cu

mu

lati

ve In

cid

en

ce

(%)

High risk atopic disease. Hydrolysate formula with GOS&FOS 8 g/L, Intervention Birth to 6 months; Follow-up to 2 years Arslanoglu S, et al. J Nutr 2008;138:1091-1095

0

5

10

15

20

25

30

Atopic Dermatitis Recurrent Wheezing Allergic Urticaria

Prebiotic Formula (GOS:FOS, 9:1), n=66

Control Formula, n=68

*p < 0.05

Prebiotics in infants for Prebiotics in infants for prevention of allergic disease prevention of allergic disease

and food hypersensitivity and food hypersensitivity

2/7 studies 2/7 studies reported ‘allergy outcome’ for 432 reported ‘allergy outcome’ for 432 infantsinfants

Infants at high risk of allergy fed hydrolyzed formula; Infants at high risk of allergy fed hydrolyzed formula; significant reduction in eczema up to 6 months of age significant reduction in eczema up to 6 months of age (2006)(2006) (RR 0.42, 95% CI 0.21, 0.84) (RR 0.42, 95% CI 0.21, 0.84)

Formula fed non-selected infants; no significant Formula fed non-selected infants; no significant difference in eczema up to 4 months of agedifference in eczema up to 4 months of age (RR 1.62, 95% CI 0.62, 4.26)(RR 1.62, 95% CI 0.62, 4.26)

Meta-analysis of the 2 studies: NS in eczemaMeta-analysis of the 2 studies: NS in eczema Analysis of 5 studies: no adverse effects on infant Analysis of 5 studies: no adverse effects on infant

growth growth

Prebiotics For Prevention of Allergic Disease and Food Hypersensitivity in Infants

Osborn DA, Sinn JK. Prebiotics in infants for prevention of allergic disease and food hypersensitivity. Cochrane Database of Systematic Reviews 2007, Issue 4.

Prebiotics in InfantsPrebiotics in Infants Softer stoolsSofter stools Increase friendly bacteriaIncrease friendly bacteria

BifidobacteriaBifidobacteria Simulate the gut flora similar to breastfed infantsSimulate the gut flora similar to breastfed infants Reduced infection rate?Reduced infection rate? Reduced allergy?Reduced allergy? More data neededMore data needed May have important effects in newbornsMay have important effects in newborns No data on efficacy in premature infantsNo data on efficacy in premature infants

SummarySummary Commensal micro-organisms are important Commensal micro-organisms are important

for neonatal healthfor neonatal health Breastfed infant modelBreastfed infant model SurrogatesSurrogates

ProbioticsProbiotics risk vs benefitsrisk vs benefits

PrebioticsPrebiotics safetysafety evidence?evidence?