Probiotics for Preterm Neonates: Challenges and Opportunities

1. Probiotics for preterm neonates –what lies ahead? Prof Sanjay Patole, MD, DCH, FRACP, MSc, DrPH Centre for Neonatal Research and Education KEM Hospital for Women, University of Western Australia, Perth

2. Routine probiotic supplementation (RPS) Reduced incidence of NEC associated with introduction of probiotics in a NICU Hoyos AH 1999 Cohort study of probiotics in a North American NICU Janvier et al. 2014 Currently 15 tertiary NICUs in Australia provide RPS for preterm VLBW neonates

3. Probiotics prevent NEC in preterm neonates 30 Trials from 17 nations (n=6655), 13 Systematic reviews NEC Stage ≥II: RR: 0.39 (95% CI=0.27-0.56), p<0.00001 All cause mortality: RR: 0.58 (95% CI=0.46-0.75), p<0.0001 NEC related mortality: RR: 0.38 (95% CI 0.18 -0.82) Time to full feeds: WMD: -1.32 (95% CI -1.48 to -1.17) Probiotics for preterm neonates: Enough is enough!! Barrington 2012

4. Probiotics: fishing in the ocean.Vandenplas 2012 • The politicsof probiotics: probiotics, NEC and the ethics of neonatal research. Janvier 2013 • Probiotics to prevent NEC- Too cheap and easy?Taylor 2014 • Myth:NEC: probiotics will end the disease.Caplan 2011 • Probiotics strain forcredibility.Hamilton-Miller 2000

5. Probiotics for preterms- what lies ahead? Challenges and opportunities

6. Extremely preterm neonates • Extremely preterm neonates are most deserving of probiotic supplementation. • Data on ELBW neonates from RCTs (N=1500) and reports on routine use of probiotics is assuring. • Probiotic sepsis is easy to treat compared with sepsis due to other organisms.

7. Benefits of probiotics in ELBW neonates may be suboptimal • Frequent exposure to antibiotics • Frequent stoppage of feeds • Recurrent episodes of late onset sepsis by CONS • Dependence on parenteral nutrition

8. Exposure to antibiotics • Early postnatal exposure to Ampicillin and gentamicin had significant adverse effects on evolution of gut flora in infants. • Antibiotic-treated infants had ↑↑ Proteobacteria (p=0.0049) and ↓↓ Actinobacteria (p=0.00001), ↓ Bifidobacterium (p=0.0132) and ↓ Lactobacillus (p=0.0182) compared with controls 4 weeks after stopping antibiotics. • Proteobacteria levels significantly higher by week 8 in the treated infants (p=0.0049). Fouhy Antimicrob Agents Chemother 2012

9. Exposure to antibiotics • Preterm neonates who received 5-7 days of empiric antibiotics in the 1st week had relative abundance of Enterobacter (p=0.016) and lower bacterial diversity in week 2 and 3. • Higher frequency of NEC, LOS, and death in those receiving early antibiotics vs those not exposed to antibiotics. Greenwood,J Pediatr 2014 Feb • Association of prolonged exposure to antibiotics with LOS, NEC and death in preterm neonates. Cotten, Kuppala, Alexander, Shah

10. Intrauterine growth restriction (IUGR) Dorling et al: Meta analysis of independent case series • 14 studies compared NEC rates in neonates who had fetal AREDF-UA with controls (forward fetal EDF). • 9 studies showed ↑odds of NEC in those with fetal AREDF. OR: 2.13 (95% CI: 1.49-3.03) ADC Fetal Neonatal Ed 2005

11. Frequent signs of feed intolerance (e.g. abdominal distension, visible ropy bowel loops, large/coloured gastric residuals) and the fear of NEC means it often takes 2-3 weeks to reach 120-150ml/kg/day feeds. • Median (IQR) time to full feeds in IUGR vs AGA extremely preterm neonates: 20 (15-34) vs. 16 (12-24) days, p=0.008 Shah et al. JMF Neonatal Med 2014 Oct

12. Kempley et al • Post-hoc analysis of data on neonates <29 weeks from a RCT (ADEPT) comparing benefits of starting feeds ‘Early’ (D2) vs ‘Late’ (D6) in preterm neonates (<35 weeks) with IUGR. • Feed increments as per the protocol should have achieved full feeds by D16 in the early and D20 in the late group. ADC Fetal Neonatal Ed 2014

13. Neonates <29 weeks achieved full feeds significantly later and had higher incidence of NEC vs those ≥29 weeks. • Median (IQR) age: 28 (22-40) vs 19 (17-23) days HR: 0.35 (95% CI: 0.3 to 0.5) • NEC: 32/83 (39%) vs 32/312 (10%) RR: 3.7 (95% CI: 2.4-5.7)

14. NEC and feed intolerance in IUGR • Fetal hypoxia and redistribution of the GI blood flow to spare the brain from hypoxic injury • Hypoxic-ischaemic injury of the gut affects development of its motor, secretory, and mucosal functions, and increases its postnatal vulnerability to ileus, altered colonization, and bacterial invasion. • Postprandial rise in SMA flow is compromised • Pseudo-obstruction due to meconium plug, ↑ LOS

15. IUGR • Significantly decreased intestinal weight and length, ileal and colonic weight/cm, and villous sizes at birth in piglets with IUGR vs same-age controls. • ↑ Markers of apoptosis and ↓ markers of proliferation D’Inca J Nutr 2010 • ↓Bioavailability of butyrate in IUGR could adversely affect colonocyte proliferation, colonic homeostasis, and reduce mucin secretion. Gaudier 2004, Barcelo 2000

16. IUGR • IUGR impairs mucus barrier development and is associated with long-term alterations of mucin expression. • Lack of an efficient colonic barrier induced by IUGR may predispose to colonic injury in neonatal as well as later life. • Continuouslyimpaired intestinal development in neonatal piglets with IUGR. Fanca-Berthon 2009, Wang 2010

17. IUGR Effect of IUGR on cecocolonic microbiota from birth to adulthood in rats with vs without IUGR • Bacterial density ↑ at D5 and ↓ at D12 in IUGR • Adult rats with IUGR had fewer Bifidobacteria at D40 and more bacteria related to Roseburia intestinalis at D100 Fanca-Berthon JPGN 2010

18. Baseline fecal Bifidobacteria in IUGR No baseline differences in the proportion of detectable B. counts between extremely preterm IUGR and AGA neonates. Probiotic: IUGR vs AGA: 7(33%) vs 22 (42%), p=0.603 Control: IUGR vs AGA: 1(6%) vs 1 (2%), p=0.429 Patole et al. PLOS ONE 2014 March (Post-hoc analysis of data on <28 week IUGR vs AGA)

19. Response to probiotic suppl. in IUGR Response to probiotic did not differ between IUGR and non-IUGR neonates (p=0.589), after adjusting for baseline counts and treatment allocation. IUGR neonates on probiotic (vs placebo) showed a non-significant trend towards a youngerpostnatal age atFEF (adjusted for age at start of MEF): Median (IQR) age: 16 (12-26) vs 19 (11-25) days

20. Probiotics can facilitate enteral nutrition • Secreted products • Products of fermentation (SCFA) • Influence on intestinal neuroendocrine factors • Gut mediators secreted as an immune reaction to probiotics Soret 2010, Barbara 2005, Cherbut 2003

21. Opportunities for advancing knowledge • Assessing nutritional benefits of probiotics is important. Jape-Athalye et al AJCN 2014 Nov. • Colonisation depends on strain properties, and host related factors such as gestational and postnatal age • Animal models: Strain selection for clinical use (Wu 2013) • Early vs Late: Highest colonization rate when the suppl. was started between 24 and 48 hours after birth. (Yamasaki 2012) • Single ve Multi-strain probiotic (Ishizeki 2013) • Live vs Inactivated/killed probiotic (Awad 2010)

22. Opportunities • Real life benefits of probiotics may not be as dramatic as reported in RCTs. • Reporting outcomes and safety data on RPS is important to know real life benefits and uncommon/rare adverse effects. • Strain specific population data for guiding clinical practice. • Assessing the economic benefits of probiotics is important. • Advances in technology: Improve tolerance of probiotic strains to bile, acid, and oxygen for enhanced benefits.

23. Challenges • Cooperation between various stakeholders is urgently required for quality control and classification of probiotics. • Field difficulties and prioritiesin resource limited set ups • Politics of probiotics • Probiotics willnot be a panacea for NEC, an illness that is known to present at different postnatal ages with different triggers and different presentations.

24. Challenges: Probiotic bacteremia/sepsis Case series of Bifidobacteriumlongumbacteremia in three preterm infants on probiotic therapy. Zbinden et al. Neonatology. 2015 Bifidobacterium longumbacteremia in preterm infants receiving probiotics. Bertelli et al. Clin Infect Dis. 2014 Fatal gastrointestinal Mucormycosis in an infant following use of contaminated ABC Dophilus powder from Solgar Inc. http://www.cdc.gov/fungal/rhizopus-investigation.html

25. Resource limited set ups Probiotic issues • Product/Strain selection, Cost, Cold storage? • Import or locally available? Quality assurance and check? • Microbiologyback up on site? Baseline data? • Priorities: VLBW, ELBW, IUGR? Hospital vs Community? Strategies for prevention of NEC • Antenatal glucocorticoids, Maternal/Donor breast milk • Avoid formula, Standardised feeding protocol • Avoid undue prolonged exposure to antibiotics

26. Probiotics for preterm neonates All good?

27. PIPs trial Multi-centre double blind randomised placebo controlled trial B. breve BBG-001 ( 2.1 to 5.3 × 108 cfu daily) in infants <31weeks Randomised before 48 hrs. Primary outcomes: NEC ≥ Bell Stage II, LOS, Death. ITT analysis adjusted for sex, gestation and randomisation within 24 hours and allowing for clustering of multiples. Costeloe et al. Arch Dis Child 2014;99: A23-A24

28. PIPs results 1310 infants randomised Median gestation 28.0 weeks, Birth weight1010g Age starting intervention 44 hours No adverse events related to the intervention No benefits in ANY of the outcomes of interest Conclusions: B. breve BBG-001 did not have any advantage Highlight need to assess the efficacy of different strains Challenges the validity of combining trials using different probiotic interventions in meta-analyses

29. Thank you!!

30. Prebiotics in preterm neonates 7 RCTs (n=417),NEC: 5 trials (n=345), LOS: 3 trials (n=295) NEC: RR: 1.24 (96% CI: 0.56-2.72) LOS: RR:0.81 (95% CI: 0.57-1.15) TFF: 3 RCTs (n=295); no improvement Bifidobacteria growth ↑↑ in prebiotic group WMD: 0.53 (95% CI: 0.33, 0.73) *106 colonies/g, p <0.00001) Reduced stool viscosity and pH No significant adverse effects Srinivasjois Clin Nutr 2013 Dec

31. Opportunities in the field of prebiotics • Large RCTs of Prebiotics vs placebo, Pro vs Synbiotic • Assess consumption of specific HMOs by different probiotic strains for developing optimal pre and probiotic combinations (Synbiotic) Garrido et al. Microbiology 2013 • Maternal vs donor breast milk HMO and secretor status

32. *Before vs AfterRPS: <33 weeks (n=834 vs 990) NEC/All cause mortality: 73 (9%) vs 52 (5%) OR: 0.57 (0.38-0.85), p=0.005 NEC (≥ Stage II): 25 (3.0%) vs 15 (1.5%) OR: 0.53 (0.27-1.01), p=0.054 All cause mortality: 56 (7%) vs 39 (4.0%) OR: 0.58 (0.37-0.91), p=0.019 Any gut perforation: 31 (3.7%) vs 15 (1.6%) *(Dec 2008-Nov 2010) vs (June 2012-May 2014) @ KEM Perth

33. Before vs AfterRPS: <28 weeks (n=250 vs 250) (1) NEC/All cause mortality: 52 (21%) vs 34 (14%) OR: 0.62 (0.37-1.02), p=0.05 (2) NEC (≥ Stage II): 16 (6%) vs 10 (4%), OR: 0.66 (0.29, 1.49), p=0.31 (3) All cause mortality: 42 (17%) vs 26 (10%) OR: 0.59 (0.33-1.03), p=0.06 (4) Any gut perforation: 22 (8.8%) vs 9 (4.1%)

35. “It can be argued that infection with lactobacilli is preferable over potential pathogens like Klebsiella, Enterobacter, or yeast.” Kliegman and Willoughby. Pediatrics 2005 “The debate may be shifted from whether it issafe to giveprobiotics to whether it is safe not to give probiotics topremature neonates.” Sanders et al, Gut Microbes 2010

36. Single vs multistrain probiotics • Colonisation of an ecosystem providing a niche for > 400 species is anticipated to be more successful with multistrain rather than monostrain probiotics. • “Given the association of development of monoflora with impending NEC, probiotics may protect VLBW neonates by enforcing diversity of flora or by preventing colonization with pathogens”. Kleigman et al. Pediatrics 2005

37. Based on the complexity of gut flora and the pathogenesis of NEC, and the multiple mechanisms of benefit of probiotic strains, multistrain probiotics may be more effective than single-strain probiotics. • Combination of probiotic strains in a product does not necessarily add to the benefits of each strain.Consensus meeting report:London, Nov 2009 • Strain combinations can beantagonistic, compatible or synergistic. Salminen et al. 2009

38. Dose • There will be an optimal dose below which benefits may not occur, as survival and proliferation to adequate numbers after overcoming the barriers (e.g. gastric acid, bile, competing pathogens), is not ensured. Lewis et al. 1998, Martin et al. 2008 • To be functional, probiotics have to be viable and in sufficient dosage levels, typically 106 to 107 cfu/g of the product. Galdeano et al. 2004, Shah et al. 2000

39. No standardised number of probiotic bacteria that would ensure an effect. • The effective quantity, for a given effect and a given strain, is the quantity which has demonstrated an effect in a clinical trial. Consensus meeting report- London, November 2009.

40. Scientific advances • Microencapsulation, improving thermal tolerance of strains • ↑ Gastric transit, GI persistence, and efficacy by cloning listerial betaine uptake system into the strain • Evaluating bile salt hydrolase to increase BA tolerance • Evaluating mucin degradation activity and translation ability • Designer (Genetically modified) probiotic strains • Metagenomics and metabonomics

41. Can more trials help? • A RCT of 2000 neonates and a baseline incidence of 8% would have to show a doubling of the incidence of NEC to overturn the benefits shown in the trials completed to date. Such a reversal of effects has never been demonstrated in clinical medicine. Barrington KJ, Arch Dis Child Educ Pract Ed 2011 • A RCT of ~ 4,500 neonates will have to show “no effect” (RR = 1.0) in mortality after probiotic supplementation.

42. Economic analyses (↓ NEC by 50%) • NEC expenses: 10 to 15 million dollars/year in Australia • Probiotic cost: $30 to $70 per baby ($5000/year) • Don’t forget the lifelong stress of parents caring for a child with NDI after severe NEC