Josef Neu, MD University of Florida, Gainesville, FL

1. Necrotizing Enterocolitis:
Why Such an Enigma
Josef Neu, M.D.
neuj@peds.ufl.edu
University of Florida

2. DISCLOSURE STATEMENT
Speaker: Josef Neu
Affiliation / Financial
Interest
Organization
Infant Microbial
Therapeutics
Research Grant
National Institutes of
Health
Research Grant (Randomized Trial of
Antibiotics and Microbiome)
Dr. Neu has disclosed the following relevant financial relationships. Any real or apparent
conflicts of interest related to the content of this presentation have been resolved.
I will not discuss any off-label use and/or
investigational use in my presentation

3. Agenda
Definition of “NEC”: Can we focus
on a “Classic NEC”?
Dysbiosis and NEC: Relation to Pro
and Anti-Inflammatory mediators.
Are neonatologists causing
dysbiosis in preterms?
Can we prevent dysbiosis?
The Future

4. A
C
B
“Classic” NEC
Neu, J. and Walker , W. A. New England Journal of Medicine, Jan. 2011

5. Neurodevelopment and NEC
Bhandari, et al. Mediators of Inflammation, 2018

6. Proposed Mechanism for
Neurodevelopmental Outcomes
Associated with NEC
Bhandari, et al. Mediators of Inflammation, 2018

7. Historical Perspective: Being led
astray: 50 years---not much
progress
•Lumping of several
diseases called
“NEC” into the same
data set.
•Animal models that
do not represent the
disease we see in
human preterms.
•Narrow focus on
individual pathways
rather than systems.

8. Gestational Age and NEC
Incidence of NEC increases by 3% for every 250 gram
decrement: Fitzgibbons, et. al, J Peds Surg. 2006.
Many NICUs “never see NEC”
NEC in babies between 1250-1500grams<1%,
But 500-750 grams is 9-12%.
If NICU A sees 10 babies <750 grams/year=1 baby with NEC.
If NICU B sees 50 babies <750 grams/year=5 X as many babies with NEC as
NICU A.

9. “Stage 1,2 and 3” NEC
Cardiogenic Ischemia
Variants of Food Protein Induced Enterocolitis
Syndrome
Spontaneous Isolated Intestinal Perforations
Misrepresentation of pneumatosis—
“poopatosis”
Placement of Drain for pneumoperitoneum
without direct visualization of bowel
Congenital bowel anomalies (e.g.,
Hirschprung’s aganglionosis)
NEC “Imposters”

10. Is there a Clear Definition of NEC?
Bells is Broken
•Stage 1-Too non-specific
and the term should not
be used.
•Stage 2-Radiographic
signs can be “fuzzy”.
•Stage 3- Free air on
radiograph could signify
intestinal necrosis or
Spontaneous Intestinal
Perforation (SIP)

11. “Poopatosis”
29 week Gestation Preterm: Abdomen Soft,
baby taking NG feeds well but Incidental
Finding on Radiograph

12. 25 week Preterm, 5 days old, advancing
enteral feedings of breast milk, on
hydrocortisone for “hypotension”---distended
abdomen.

14. Spontaneous Intestinal
Perforation vs. NEC

15. “NEC” or “FPIES”??

16. A
C
B
“Classic” NEC
Neu, J. and Walker , W. A. New England Journal of Medicine, Jan. 2011

17. What Causes Classic “NEC”?
Pathophysiology
Where’s Hypoxia-Ischemia
and Feeding?

19. Mean Gestational Age at NEC
Diagnosis
Pammi, M. et al. Microbiome, 2017
23 week preterm
29 week preterm

20. Mean Gestational Age at NEC
Diagnosis
Pammi, M. et al. Microbiome. 2017 Mar 9;5(1):31
• Microvasculature Changes?
• Immature Barrier
• TLR Developmental Pattern?
• Microbiota changes?

21. Development of Intestinal
Angiogenesis With Microbes
• Villi from (P14) versus (P28)
conventionally raised mice.
• Capillary networks are
stained green (FITC).
Stappenback, Hooper and Gordon, PNAS, 2002

22. Development of Intestinal
Angiogenesis With Microbes
Stappenback, Hooper and Gordon, PNAS, 2002

23. The Intestinal Barrier: Cells
Abreu, M. Nature Immunology Feb. 2010

25. From Madara J. Building an intestine – architectural contributions of
commensal bacteria. New Engl. J. Med. 2004; 351: 1685-86.

26. Lesson
Low grade stimulation (“tickling”) of toll receptors can
prevent high grade inflammation and intestinal
damage and promotes intestinal homeostasis.

27. FECAL MICROBIOTA: NEC
Mai V, Young C. PLOS One, May 2011
• Proportions of the four major phyla two weeks before and the week of
diagnosis

28. Microbial Shift Prior to NEC
From Claud, E. et al . Microbiome, 2013

29. Abundance of Gamma-
Proteobacteria
Warner, B. et al. Lancet March 8,2016

31. Comparison of three major phyla:
Proteobacteria, Firmicutes and Bacteroidetes
Phylum Gram Staining Functional Relationship Comment
Proteobac
teria
Gram
negative
High Lipopolysaccharide (LPS) content
in cell wall. Abundance of
Proteobacteria increased prior to
exacerbations of inflammatory bowel
diesease. Strong stimulator of TLR4. E.
Coli, Klebsiella and Pseudomonas are
representatives.

32. Comparison of three major phyla:
Proteobacteria, Firmicutes and
Bacteroidetes
Phylum Gram Staining Functional Relationship Comment
Firmicutes Gram positive Lactobacilli are a common class of the Firmicutes
phylum. Have high lipoteichoic acid in the cell wall,
but low LPS. Have excellent capacity for energy
harvest. Produce butyrate in high quantities. Butyrate
is a major fuel for colonocytes and important for
maintenance of tight junctions.

33. Comparison of three major phyla:
Proteobacteria, Firmicutes and
Bacteroidetes
Phylum Gram Staining Functional Relationship Comment
Bacteroidetes Gram
negative,
anaerobic, rod
shaped
bacteria.
Involved in fermentation of carbohydrates
(propionate and acetate producers), utilization of
nitrogenous substances, and biotransformation of bile
acids. Bacteroides fragilis is a representative. The
immunomodulatory molecule, polysaccharide A
(PSA), of B. fragilis mediates the conversion of CD4+ T
cells into Foxp3+ Treg cells that produce IL-10 during
commensal colonization. PSA is not only able to
prevent, but also cure experimental colitis in animals.
Propionic acid is also a strong inducer of the Foxp3+ R
regulatory pathway.

34. Causes of Inappropriate
Colonization “DYSBIOSIS”
Type of Diet:
Human Milk
versus
Formula
Lack of Enteral
Feeding; TPN,
Intestinal pH
Antibiotics
and Microbial
Environment
Do Common Neonatal Practices
Cause NEC?

35. Early Antibiotic
or Acid
Suppressor
Use: What are
we really
doing?

36. Early
Antibiotic
Use:
What are
we really
doing?
Antibiotic Induced Dysbiosis
Preidis and Versalovic, Gastroenterology
2009;136:2015-2031

37. “ So I give a couple days antibiotics to my
preterm patients, what does that matter if I
change the microbes in the GI tract since I
could potentially be saving the baby’s life by
treating unrecognized early onset sepsis”---
Anonymous Neonatologist.

39. Most Commonly used Drugs in the NICU: Majority of
VLBW infants are Exposed to Antibiotics
Top 10 Medications Prescribed in the NICU

40. Odds Ratio of NEC
with Increased Days on Antibiotics
Alexander, V.N. J. Pediatrics, Sept. 2011
Average length of
Treatment increases
odds by
50%

41. Pediatrics, 2012, 129. e-40-45
Gastric Acid Inhibition

42. Effect of H2 Blocker on
Microbiome
Proteobacteria
Firmicutes
Gupta RW, et al., JPGN, 2013

43. Things we do to Mess Things
Up: Lack of Enteral Feeding
• Excuses To Withhold ENTERAL “Feedings
Low APGAR scores.
Umbilical catheters.
Apnea and Bradycardia.
Mechanical ventilation.
CPAP.
Vasoactive drugs.
TPN is available.

44. Dr. Elsie Widdowson (1906-
2000)
The suckled pig’s
duodenum gains 42% of
it’s weight in the first 24
hours after birth.
Ashwell M
Nature 406, 844 (24 August 2000)

45. Demehri, FR., et al. Cellular and Infection Microbiology, Dec. 2013
Effect of Total Parenteral
Nutrition (TPN) in Mice

46. Morbidities: Early vs. Late
Feeding
Konnikova, et al. PLOS One 2015

48. NEC: A Diagnostic Dilemma

49. Marker Cutoff Point Sensitivity Specificity LR+ LR- AUC
(95%CI)
P
I-FABP 2.25 pg/mmole
creatinine
0.93 0.90 9.3 0.08 0.98 (0.94-
1.0)
<0.001
Claudin-3 8OO.8 INT 0.71 0.81 3.74 0.36 0.76 (0.59-
0.94)
0.016
Calprotectin 286.2
microgram/gram
feces
0.86 0.93 12.29 0.15 0.94 (0.85-
1.0)
0.001
NEC versus Non NEC
Differentiation
Thuijls, et al. Annals of Surgery, 251 (6), June 2010

51. Routine Use of Probiotics

52. Deshpande, G. Pediatrics, 2010
Meta-Analysis -NEC

53. Summary of 2010 Meta Analysis
• 11 studies evaluated.
• 10 different probiotic preparations.
• Risk for NEC and death significantly lower in probiotic group.
• Sepsis did not differ.
• “Overall evidence indicate that additional placebo controlled trials
are unnecessary if a suitable probiotic product is available”.
• Commentary: Is it ethical to not use probiotics in
preterm infants?
Deshpande,G. Pediatrics, May
2010

54. J Pediatr. 2011 Apr;158(4):672-4.

55. ProPrems Australia
Jacobs, et al. Pediatrics, 2013.
• 1099 VLBW infants randomized to receive probiotic combination (not
powered from NEC---primary outcome late onset sepsis).
• No difference in sepsis or all cause mortality
• NEC went from 4.4 to 2.0 (secibdary analysis), p=0.03: Number
needed to treat=43, 95% confidence interval 23-333.
• No effect on NEC in babies < 1000 grams Birth Weight.

56. Largest Study so Far: UK Pips
Trial (Costello, et al. Lancet Feb., 2016)
• Double Blinded, randomized, Prospective.
• Bifidobacterium breve probiotic
• 23-31 weeks gestational age enrolled
• Powered for NEC as primary outcome: 1315 infants
enrolled
• Results: NO differences in:
• NEC
• Late Onset Sepsis
• Death

57. Fatal gastrointestinal mucormycosis in an
infant following use of contaminated ABC
Dophilus from Solgar Company
CDC, FDA, and the Connecticut Departments
of Public Health and Consumer Protection,
are investigating a fatal case of GI
mucormycosis in a premature infant of 29
weeks gestation following the use of a
probiotic supplement called ABC Dophilus
distributed by Solgar, Inc., Leonia, NJ.
FDA ALERT!!
https://www.cdc.gov/mmwr/preview/mmwr
html/mm6406a6.htm

58. Food Supplement or Drug?
• It depends!
• Medical claim (e.g. prevention of NEC, treatment of diarrhea) usually
should be considered a drug.
• Drugs that are sold by prescription are subjected to rigorous testing.
• Foods can be sold by anyone and not subjected to rigorous
standards.

59. • There are also no current standards for
“quality control of a reconstituted product”
and good manufacturing practices for use of
probiotics as drugs are not available.
 Quality of products questionable—amount
provided, strain specificity questionable.
 Use of “off the shelf” products!
Current Status in U.S.

60. Viswanathan, V. et al J. Perinatology, 2016
No Evidence for Safety or
Efficacy for 90% of Probiotics
used in U.S. NICUs

61. Kane, A. J. Peds, 2018
Routine Supplementation of LGG

63. “Is it ethical to not
tell parents about
probiotics and the
prevention of NEC
in preterm
infants?”

64. Microbes in Human Milk
• Concern about pathogens has
led to pasteurization of donor
milk.
• Studies using both culture
based and non culture based
techniques show that there are
endogenous microbes in human
milk.

65. Microbial Dose from Human Milk
•Assume intake of 800 ml/day
•Assume 105-6 bacterial cells/ml
•This will provide 10 7-8 bacterial
cells (personalized?) daily, close
to the dose in most probiotic
studies.

66. Summary and the
Future
• NEC Pathogenesis is Multifactorial (Even if we invoke a “Classic
NEC”). We need better definitions.
• Treatment of NEC once the disease occurs is extremely difficult. We
need to prevent.
• The intestinal microbial environment along with developmental
aspects of the GI tract are key in understanding the pathogenesis of
“classic” NEC.
• We need to have better systems (enteroids, animal models) to
evaluate mechanisms that fulfill criteria for causality derived from
strong associations found in humans.
• Once we have clear understanding of the causes of the different
forms of “NEC”, we will be best able to target preventative strategies.