Probiotics – PrebioticsNovel Strategies That May Prevent Neonatal Disease by Richard J. Schanler, M.D. Schneider Children’s Hospital at North Shore, Manhasset, NY and Albert Einstein College of Medicine, Bronx, NY schanler@nshs.edu

1. Probiotics – Prebiotics
Novel 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, NY
schanler@nshs.edu
February 2010

2. 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 speciesmicroorganisms, 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 injuryActivate toll-like receptors to protect against gut injury
10 trillion10 trillion

3. 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

4. Some of the Intestinal MicrofloraSome of the Intestinal Microflora
Identified in Infants are BeneficialIdentified in Infants are Beneficial
 BifidobacteriumBifidobacterium
 LactobacillusLactobacillus
 StaphylococcusStaphylococcus
 StreptococcusStreptococcus
 BacteroidesBacteroides
 ClostridiumClostridium
 EscherichiaEscherichia
 EnterococcusEnterococcus
 VellonellaVellonella
 PropionibacteriumPropionibacterium
 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

5. 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”

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

7. Carbohydrate FermentationCarbohydrate Fermentation
 Major function of intestinal bacteriaMajor function of intestinal bacteria
 Bifidobacteria and lactobacilliBifidobacteria and lactobacilli
 ferment carbohydrate to produce lactic acidferment carbohydrate to produce lactic acid
 Acidic milieu potentiates nonpathogenicAcidic milieu potentiates nonpathogenic
bacteriabacteria
 ColiformsColiforms
 ferment carbohydrate to COferment carbohydrate to CO22 and waterand water
 neutral pHneutral pH

8. Stool pH:
Indicator of Normal Gut Fermentation
4.8
5
5.2
5.4
5.6
5.8
6
3rd Day 1st
Month
2nd
Month
3rd
Month
4th
Month
Breastfed
Formula-Fed
Indrio et al. Pediatr Res 2007; 62:98

9. Approaches to Create IntestinalApproaches to Create Intestinal
Microflora Like Breastfed InfantMicroflora Like Breastfed Infant
Two possible approaches:Two possible approaches:
1.1. ProbioticsProbiotics
 Feed live microorganismsFeed live microorganisms
2.2. 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.

10. ProbioticsProbiotics

11. ProbioticsProbiotics
 1900s Elie Metchnikoff, Pasteur Institute1900s Elie Metchnikoff, Pasteur Institute
 ““Long life span of Bulgarian peasantsLong life span of Bulgarian peasants
due to ingestion of fermented milksdue to ingestion of fermented milks
containing large numbers ofcontaining large numbers of
nonpathogenic bacteria”nonpathogenic bacteria”

12. 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

13. 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

14. Probiotics Enhance Host DefenseProbiotics Enhance Host Defense
 Prevent colonization by pathogenic organismsPrevent colonization by pathogenic organisms
 Competition for nutrientsCompetition for nutrients
 Competition for attachment sitesCompetition for attachment sites
 Produce antibacterial compounds unfavorable toProduce antibacterial compounds unfavorable to
pathogenspathogens
 Volatile fatty acidsVolatile fatty acids
 Modified bile acidsModified bile acids
 Recruit immune cellsRecruit immune cells
 Activate immune and inflammatory responsesActivate immune and inflammatory responses

15. 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 barrierLarge surface barrier
 Gut permeabilityGut permeability
 TranslocationTranslocation
 Passage of intestinal bacteria through intestinal mucosaPassage of intestinal bacteria through intestinal mucosa
 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

16. 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

17. 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-10IL-10
 ↓↓ ubiquinationubiquination
 ↓↓ IL-8IL-8
 ↓↓ tumor necrosis factor (TNF)tumor necrosis factor (TNF)
 InflammationInflammation
 ↑↑ IgAIgA
 ↑↑ IL-6IL-6
 ↑↑ NK call activityNK call activity
 ↑↑ phagocytosisphagocytosis
 ↑↑ ββ-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-

18. Probiotics and Antibiotic-Associated DiarrheaProbiotics and Antibiotic-Associated Diarrhea
 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 x10Lactobacillus GG, 1-2 x1099
 PlaceboPlacebo
 Diarrhea incidenceDiarrhea incidence
 Lactobacillus: 7 cases (8%)Lactobacillus: 7 cases (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

19. Probiotics Reduce Diarrhea in Children:Probiotics Reduce Diarrhea in Children:
Five Meta-AnalysesFive Meta-Analyses
No. trialsNo. trials No. subjectsNo. subjects 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 dduration: 1.1 d
1
H. Szajewska & J. Mrukowicz JPGN 2001;33:S17-S25
2
C. Van Niel et al. Pediatrics 2002;109:S678-684
3
J. Huang et al. Dig Dis Sci 2002;1:2626-2634
4
H. Szajewska et al. APT 2007;25:257-264
5
H. Szajewska et al. APT 2007;25:871-881

20. Necrotizing EnterocolitisNecrotizing Enterocolitis
 GI catastropheGI catastrophe
 PrematurityPrematurity
 Bacterial floraBacterial flora
 Reduction in commensal bacteriaReduction 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

21. Rationale for Probiotics in NECRationale for Probiotics in NEC
 Lactobacilli and Bifidobacteria have low rates ofLactobacilli and Bifidobacteria have low rates of
spontaneous colonization in breastfed infants <1500 gspontaneous colonization in breastfed infants <1500 g
 Probiotics reduce pathogenic bacterial colonizationProbiotics reduce pathogenic bacterial colonization
 Multiple courses of antibioticsMultiple courses of antibiotics
 Abnormal NICU floraAbnormal NICU flora
 Microbial etiologies in NECMicrobial etiologies in NEC
 Reinforce intestinal barrierReinforce intestinal barrier
 Prevent bacterial translocationPrevent bacterial translocation
 Alleviate intestinal inflammationAlleviate intestinal inflammation
 Balance of anti- and pro-inflammatory cytokinesBalance of anti- and pro-inflammatory cytokines

22. Probiotics and NEC - Animal StudiesProbiotics and NEC - Animal Studies
 Bifidobacteria vs controlBifidobacteria 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 probioticsReduction in NEC with probiotics

Bifidobacteria 30%Bifidobacteria 30%

Control 70%Control 70%

23. Probiotics and Premies – JapanProbiotics and Premies – Japan
 Randomized, controlled trial, JapanRandomized, controlled trial, Japan
 VLBW infants, N=91VLBW infants, N=91
 Bifidobacterium breveBifidobacterium 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

24.  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

25.  ResultsResults
 NEC incidenceNEC incidence

Probiotic year: 37/1237Probiotic year: 37/1237 →→ 3%3%

Historical control year: 85/1282Historical control year: 85/1282 →→ 6.6%6.6%
 NEC mortality – significant differenceNEC mortality – significant difference

Probiotic yearProbiotic year →→ 14 cases14 cases

Historical control yearHistorical control year →→ 35 cases35 cases
Probiotics and Premies - BogotaProbiotics and Premies - Bogota
Hoyos, Int J Inf Dis 1999; 3:197Hoyos, Int J Inf Dis 1999; 3:197

26. 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 milkMean BW 1335 g, GA 31 wks; 64% rec’d human milk
 Lactobacillus GGLactobacillus GG supplementsupplement (Dicoflor, Dicofarm, IT)(Dicoflor, Dicofarm, IT) vs controlvs control
 Once daily (6 x 10Once daily (6 x 1099
) from onset feeding to discharge) from onset feeding to discharge
 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

27. Probiotics and Premies - IsraelProbiotics and Premies - Israel
 Randomized, double-blind studyRandomized, double-blind study
 Daily supplement, first feed to 36 wk corrected ageDaily supplement, first feed to 36 wk corrected age
 ABC Dophilus (Solgar, Israel)ABC Dophilus (Solgar, Israel)

Bifidobacteria infantisBifidobacteria infantis 0.35 x 100.35 x 1099
cfu/daycfu/day

Bifidobacteria bifidusBifidobacteria bifidus 0.35 x 100.35 x 1099
cfu/daycfu/day

Strep thermophilusStrep thermophilus 0.35 x 100.35 x 1099
cfu/daycfu/day
 Power analysisPower analysis
 n=145 for reduction in NEC from 15% to 5%n=145 for reduction in NEC from 15% to 5%
Bin-Nun et al, J Pediatr 2005;147:192-6

28. Probiotics ControlProbiotics Control
Probiotics and Premies - IsraelProbiotics and Premies - Israel
Birth weight (g)Birth weight (g) 11521152 11111111
Gestation (wk)Gestation (wk) 29.829.8 29.329.3
SGA (%)SGA (%) 2525 1515
Sepsis (%)Sepsis (%) 4343 3333 nsns
Antibiotics (d)Antibiotics (d) 12.512.5 14.914.9
Full feeding (d)Full feeding (d) 14.614.6 17.517.5
TPN discontinued (d)TPN discontinued (d) 16.616.6 18.618.6
Human milk exclusive (%)Human milk exclusive (%) 5858 6464
Partial human milk (%)Partial human milk (%) 1717 1212
Formula (%)Formula (%) 2525 2323
NPO (d)NPO (d) 5.25.2 4.84.8
All differences are not significant Bin-Nun et al, J Pediatr 2005;147:192-6

29. Probiotics, Premies, NEC - IsraelProbiotics, Premies, NEC - Israel
Probiotics ControlProbiotics Control P valueP value
††
Bell stage of NEC 1-3 All differences are significantBell 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 2.32.3 0.0050.005
NEC or death (n, %)NEC or death (n, %) 6 (8%)6 (8%) 17 (23%)17 (23%) 0.0250.025

30. Probiotics, Premies, & NEC - TaiwanProbiotics, Premies, & NEC - Taiwan
 Randomized, controlled, blinded trial, infants <1500 gramsRandomized, controlled, blinded trial, infants <1500 grams
 Mean 1100 g, ~28 wksMean 1100 g, ~28 wks
 Probiotic: InfloranProbiotic: InfloranTMTM
with breastmilk twice daily, n=180with breastmilk twice daily, n=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 milkBreastmilk: either mother’s own or donor human milk
Lin et al, Pediatrics 2005;115:1-4

31. Probiotics, Premies, & NEC - TaiwanProbiotics, Premies, & NEC - Taiwan
Probiotics ControlProbiotics Control PP 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

32. Probiotics, Premies, & NEC – Taiwan IIProbiotics, Premies, & NEC – Taiwan II
 Randomized, controlled, masked trial, infants 500-1500 gRandomized, controlled, masked trial, infants 500-1500 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 wkwith milk twice daily x 6 wk
 Lactobacillus acidophilusLactobacillus acidophilus 101099
 Bifidobacteria infantisBifidobacteria infantis 101099
 Control groupControl group
Lin H-C et al, Pediatrics 2008;122:693-700

33. Probiotics, Premies, & NEC – Taiwan IIProbiotics, Premies, & NEC – Taiwan II
Probiotics ControlProbiotics Control PP valuevalue
Birth weight (g)Birth weight (g) 10291029 ± 246± 246 10771077 ±± 214214 .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, stageNEC, 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

34. Probiotics and Time to Full FeedingsProbiotics and Time to Full Feedings
WMD = weighted mean difference, probiotic - control
Probiotic therapy shortens time to full feeding, P = 0.02
Deshpande G, Rao S, Patole S. Lancet 2007; 369:1614-1620
ProbioticsProbiotics ControlControl WMD (95% CI)WMD (95% CI)
Kitajima 2007Kitajima 2007 4545 1717 ±± 8.48.4 4646 20.5 ± 11.920.5 ± 11.9 -3.9 (-7.7, 0.7)-3.9 (-7.7, 0.7)
Bin Nun 2005Bin Nun 2005 7272 14.614.6 ± 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 1515 ±± 88 4141 1717 ±± 99 -2 (-5.7, 1.7)-2 (-5.7, 1.7)
TOTALTOTAL 156156 160160 -2.7 (-5, -0.5)-2.7 (-5, -0.5)

35. Probiotics, Premies, & NECProbiotics, Premies, & NEC
 BogotaBogota 6.66.6 →→ 3%3%
 Lactobacillus acidophilusLactobacillus acidophilus
 Bifidobacteria infantisBifidobacteria infantis
 ItalyItaly 2.72.7 →→ 1.4% ns1.4% ns
 Lactobacillus GGLactobacillus GG
 TaiwanTaiwan 5.35.3 →→ 1.1%1.1%
6.56.5 →→ 1.8%1.8%
 Lactobacillus acidophilusLactobacillus acidophilus
 Bifidobacteria infantisBifidobacteria infantis
 IsraelIsrael 1616 →→ 4 %4 %
 Bifidobacteria infantisBifidobacteria infantis
 Bifidobacteria bifidusBifidobacteria bifidus
 Streptococcus thermophilusStreptococcus thermophilus
Randomized trials

36. Meta-Analysis Probiotics & NECMeta-Analysis Probiotics & NEC
ProbioticsProbiotics ControlControl RR (95% CI)RR (95% CI)
NECNEC >> Stage IIStage II 13/63713/637 37/62737/627 0.32 (0.17, 0.60)0.32 (0.17, 0.60)
All MortalityAll Mortality 16/60116/601 39/60639/606 0.43 (0.25, 0.75)0.43 (0.25, 0.75)
NEC-Related Mortality*NEC-Related Mortality* 0/3670/367 5/3635/363 0.17 (0.02, 1.37)0.17 (0.02, 1.37)
SepsisSepsis 61/64761/647 97/63797/637 0.93 (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

37. Probiotics & MortalityProbiotics & Mortality
RR = relative risk
Probiotic group had lower overall mortality, p = 0.0007
Deshpande G, Rao S, Patole S. Lancet 2007; 369:1614-1620
ProbioticsProbiotics ControlControl RR (95% CI)RR (95% CI)
Kitajima 1997Kitajima 1997 0/450/45 2/462/46 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 59/63759/637 0.47 (0.30, 0.73)0.47 (0.30, 0.73)
Lin 2008Lin 2008 2/2172/217 9/2179/217

38. Proposed Strategies for Preventing NECProposed Strategies for Preventing NEC
 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
 ProbioticsProbiotics
 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 TreatNumber Needed To Treat
Adapted from Bell, Pediatrics 2005;115:173
24

39. 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

40. Potential ComplicationsPotential Complications
 InfectionInfection
 SepsisSepsis
 MeningitisMeningitis
 Soft tissueSoft tissue
 EndocarditisEndocarditis
 Dental cariesDental caries
 Acquired bacterial resistanceAcquired bacterial resistance

41. LactobacillusLactobacillus SepsisSepsis
 Compromised infant hostsCompromised infant hosts
 Multiple courses of antibioticsMultiple courses of antibiotics
 A. Developed secondary diarrhea, treated withA. Developed secondary diarrhea, treated with Lactobacillus GGLactobacillus GG
 Within 3 weeks of continued treatmentWithin 3 weeks of continued treatment

patients developed feverpatients developed fever

systemic symptoms of sepsissystemic symptoms of sepsis

gram positive anaerobic non-spore forming rodsgram 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;115:178
Thompson, J Perinatol 201;21:258

42. Boyle, et al. AJCN, 2005

43. 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

44. PrebioticsPrebiotics

45. 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 probioticsAct 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

46. Oligosaccharides: 3rd
Most Common Component
in Mature Human Milk
0
10
20
30
40
50
60
70
Lactose
Fat
Oligosaccharides
Protein
Upper Range of Concentration
Lower Range of Concentration
g/L
Adapted from Kunz et al, Clin Perinatol 1999;26:307

47. Human Milk OligosaccharidesHuman Milk Oligosaccharides
 Colostrum – highest concentrationColostrum – highest concentration
 Mature milk – lower contentMature milk – lower content
 Contain galactose, glucose, N-acetylglucosamine, fucose,Contain galactose, glucose, N-acetylglucosamine, fucose,
sialic acidsialic 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

48. Human Milk OligosaccharidesHuman Milk Oligosaccharides
 Receptor analogues for glycoconjugates on epithelial andReceptor analogues for glycoconjugates on epithelial and
endothelial cell surfacesendothelial cell 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
 HMO found in infant stoolsHMO found in infant stools
 Fermentation productsFermentation products
Kunz 1999, Newburg 2000, Coppa 1997, Brand-Muller 1998

49. Currently Available PrebioticsCurrently 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

50. 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

51. Stool Microflora After 6 wks Feeding Formula
with/without Oligosaccharides
BIF EC CLOS ENT
%
0
10
20
30
40
50
60
70
Formula without Oligosaccharides
Knoll, 2003; microflora by FISH analyses; Oligo supplement was FOS and GOS
BIF=Bifidobacteria, EC=E. coli, CLOS=Clostridium, ENT=Enterobacteria
**All differences significant, p < 0.05
Formula with Oligosaccharides
* *
*
*

52. Effect of Prebiotics on Stool pH
Breastfed 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 study
Differences in pH: Prebiotic vs Control, p < 0.05

53. Prebiotics Increase Fecal IgA
Breastfed Prebiotic Control Formula
µg/g feces
0
1000
Scholtens, J Nutr 2008; 138:1141 significant differences at 26 wks of study
*Prebiotic vs Control, p < 0.05
*

54. 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

55. 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
Overal
Infections
URTI Infections
treated with
antibiotics
Fever
episodes
* *
*p < 0.01
Number
of
Episode
s 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

56. GOS/FOS (9:1) Decreased Incidence of
Allergic Manifestations
*
*
*
CumulativeIncidence(%)
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

57.  2/7 studies2/7 studies →→ reported ‘allergy outcome’ for 432 infantsreported ‘allergy outcome’ for 432 infants
 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 (2006)significant reduction in eczema up to 6 months of age (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 difference inFormula fed non-selected infants; no significant difference in
eczema up to 4 months of ageeczema 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 growthAnalysis of 5 studies: no adverse effects on infant 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.

58. 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

59. SummarySummary
 Commensal micro-organisms are important forCommensal micro-organisms are important for
neonatal healthneonatal health
 Breastfed infant modelBreastfed infant model
 SurrogatesSurrogates

ProbioticsProbiotics

risk vs benefitsrisk vs benefits

PrebioticsPrebiotics

safetysafety

evidence?evidence?