The pathophysiology of necrotizing enterocolitis (NEC) has not been clearly elucidated, but recent studies support the role of unbalanced pro-inflammatory signaling, leading to intestinal necrosis in premature infants. Although breast milk feeding is thought to reduce the risk of this condition, there are no preventive or therapeutic approaches that have consistently shown to be effective for this common and devastating disease. Recent studies show that probiotic colonization is abnormal in preterm neonates, and enteral supplementation with a variety of probiotic organisms can reduce the risk of disease. This chapter summarizes the current state-of-the-art regarding probiotics and NEC, but suggests caution until appropriately regulated products are available for use in this high-risk population.
Necrotizing enterocolitis (NEC) is an inflammatory necrosis of the intestine that results in significant morbidity and mortality in premature infants.1 The disease typically has an acute onset, with symptoms and signs that include abdominal distention, emesis, bloody stools, apnea and bradycardia, and lethargy, and the diagnosis is made either by radiological evidence of pneumatosis intestinalis or surgical confirmation of the disease. There are no specific treatment options; current therapy consists of supportive care with gastric decompression, antibiotics, blood pressure support and surgery if there is clear evidence of intestinal perforation or worsening intestinal necrosis. Earlier reports suggest that peritoneal drain placement in the acute setting of NEC is as effective as invasive laparotomy on the important outcomes of survival or short-term complications of bowel function and need for intravenous nutrition.2 There are no clearly proven preventive strategies, although breast milk feeding seems to reduce the risk of disease compared with formula supplementation.3
Epidemiological investigations have suggested a significant variability in the prevalence of the disease. Reports from the United States show that the incidence ranges from 2 to 22% in neonates born weighing less than 1500 g,4 whereas international studies have identified rates in similar patients of 28% in Hong Kong,5 14% in Argentina,6 7% in Austria7 and as low as 1.5% in Japan.8 Some reports suggest a higher risk in African-Americans and boys; there seems to be only a slightly higher risk in twins and triplets of index cases.
Despite advances in neonatal care, the mortality rate following NEC has not changed significantly in the past 30 years and remains between 20 and 30%.9 There is significant short-term morbidity, including abnormal bowel function, prolonged intravenous feeding requiring central line placement, and higher costs and longer lengths of stay compared with gestational age-matched controls. In one report, surgical NEC patients resulted in higher charges by $200 000 per patient compared with non-NEC controls, a significant number from a public health perspective.10 Most concerning is the recent evidence that documents significantly higher rates of neurodevelopmental impairment in patients with surgical NEC compared with birthweight controls; as high as 59% in one recent analysis.11 Nonetheless, the pathophysiology of NEC remains controversial, and as such, specific intervention or prevention strategies are lacking. In this review, the pathophysiology of NEC will be discussed and new information provided to assess the possible role of probiotics and prebiotics in the prevention of this important and devastating disease.
Pathophysiology of NEC
The pathophysiology of NEC has not been clearly elucidated. Textbook chapters have suggested that a combination of risk factors, including prematurity, formula feeding, intestinal ischemia/hypoxia and bacterial colonization, contributes to the final common pathway; this has been referred to as the multifactorial theory. It has been shown in experimental systems that feeding, intestinal ischemia and bacteria can cause mucosal injury that can be characterized by increased mucosal permeability, interference of epithelial cell tight junctions, epithelial cell apoptosis (programmed cell death) and/or activation of pro-inflammatory signaling.12 Protection from mucosal injury is provided by several host defense factors, including mucin, trefoil factor, diverse pathogen-sensing oligosaccharides, defensin proteins, inflammatory cells, immunoglobulin and many others.13 In the neonatal animal and the preterm infant, there is evidence of increased mucosal injury and reduced host defense. In this situation, unbalanced activation of pro-inflammatory signaling would ensue with increased signal transduction, production of additional pro-inflammatory molecules and, in exaggerated cases, neonatal NEC.
Many pro- and anti-inflammatory mediators are associated with NEC based on animal or human studies. Pro-inflammatory compounds that are upregulated in NEC include tumor necrosis factor, platelet activating factor (PAF), nitric oxide, interleukin (IL)-1, IL-6, IL-18, endothelin-1, leukotrienes, thromboxanes and oxygen-free radicals.14 Several anti-inflammatory compounds downregulate intestinal inflammation, and these include prostacyclin, nitric oxide, multiple growth factors, such as transforming growth factor, epidermal growth factor, heparin-binding epidermal growth factor-like growth factor, insulin-like growth factor and erythropoietin, IL-10, IL-11, IL-12, glutamine and arginine.15, 16 It is difficult to delineate particular importance for each of these mediators, but it is likely that the balance between pro- and anti-inflammatory regulation contributes to the final common pathway of the disease.
Studies from our laboratory have documented the role of PAF in neonatal NEC in humans and animal models. PAF is an endogenous phospholipid mediator present in most cells and tissues, and is present in the intestinal mucosal environment following synthesis and secretion from epithelial cells, inflammatory cells and bacteria.17 Neonates have an impaired capacity to degrade this potent biological mediator owing to the reduced activity of the PAF-specific degrading enzyme, PAF-acetylhydrolase (Figure 1).18 PAF activates cells and downstream signal transduction after binding to the ubiquitous PAF receptor, which has been shown to be expressed in highest amounts on intestinal epithelial cells.19 It is interesting to note that newborn animals stressed with formula feeding and asphyxia express higher concentrations of PAF receptor than mother-fed controls. Following PAF receptor activation, epithelial cells have accelerated apoptosis, increased mucosal permeability and alterations in tight junctional integrity, increased signal transduction and increased expression of a variety of pro-inflammatory mediators.20 It is interesting to note that PAF appears to activate the expression of an important bacterial recognition receptor, toll-like receptor (TLR)4.
The human TLRs are a family of pattern recognition receptors that transduce signals from specific ligand components of pathogenic organisms.21 For example, lipopolysaccharide from Gram-negative bacteria binds to and activates TLR4, which is present on the surface of many different cells and tissues. After lipopolysaccharide binding to TLR4, a series of chaperone and signal transduction molecules are activated, which ultimately leads to nuclear factor-kB translocation from cytoplasm to the nucleus where this important transcription factor activates the gene expression of multiple pro-inflammatory cytokines.22 In this way, the host recognizes the pathogen and initiates steps to eradicate its presence. It is interesting to note that TLR4 is normally minimally expressed on intestinal epithelium in adults, and therefore, bacterial products do not activate an inflammatory state. In the stressed neonatal animal (and probably in humans), epithelial TLR4 expression on intestine is increased, with activation of downstream pro-inflammatory cytokines.23 It is thought that these factors contribute to NEC, and recent experiments have shown that mice with a mutation in their TLR4 gene are resistant to NEC.24
Probiotics and their role in NEC prevention
Probiotics are microbial strains of human origin, non-pathogenic, adherent to gut epithelium, colonize the intestinal tract, produce antimicrobial substances and modulate immune responses.25 These commensal bacteria are used throughout the world for infectious and antibiotic-associated diarrhea, pouchitis, inflammatory bowel disease, functional bowel disease and many other conditions. In healthy children and adults, probiotic bacteria are the most prevalent organisms in the normal intestinal flora, and studies have shown a rich diversity in the various species and strains.26 The most common commensals are Bifidobacteria and Lactobacillus species, but others include Streptococcus thermophilus, Saccharomyces strains and Escherichia coli Nissle. Studies in healthy newborn infants show the presence of multiple probiotic strains with high concentrations, but it is of interest that premature neonates appear to be quite deficient in probiotic intestinal colonization. One recent study showed that only 1 out of 29 ELBW infants was colonized with probiotic organisms and that antibiotic exposure reduced the likelihood of colonization.27 As it is hypothesized that probiotics act to downregulate pathogenic organisms and protect against intestinal inflammation, the lack of adequate colonization in the preterm neonate could place them at an increased risk for neonatal NEC. Therefore, several clinical studies have been performed in premature infants to evaluate the safety and efficacy of probiotic supplementation.
Many reports describe probiotic supplementation in preterm infants, and initial reports have shown varying rates of colonization and feeding tolerance. As different probiotic strains have different biological effects, it is difficult to extrapolate results from one unique strain to the next, and unfortunately, there is little consistency from study to study. Nonetheless, four interesting trials have been published that assess the efficacy of probiotics in the prevention of NEC (Table 1). In the first report, using historical controls as a comparison group, Hoyos treated 1282 neonates with Infloran (Bifidobacteria infantis and Lactobacillus acidophilus) and showed a reduction in NEC from 6.6 to 3.0% in the controls.28 A prospective, randomized trial conducted in 14 Italian neonatal intensive care units with 295 neonates treated with Lactobacillus GG (290 placebo controls) found no statistical difference in the incidence of NEC (2.7% control vs 1.4% L. GG), although the baseline NEC incidence was significantly lower than that expected based on the pre-trial power calculation.29 A third trial looked at the effect of Infloran in Taiwan in a prospective, placebo-controlled trial and found a significant reduction in NEC incidence with probiotics (1.1%) compared with controls (5.3%).30 The last trial published from Israel used a combination called ABCdophilus that contains B. infantis, Bifidobacteria bifidum and S. thermophilus and found a reduced incidence of NEC from 14 to 1% in babies born weighing less than 1500 g.31 These studies in total suggest a significant reduction in NEC with prophylactic probiotic supplementation, and a recent meta-analysis confirmed this result and recommended use particularly in the 1000- to 1500-g cohort.32
Some physicians and scientists favor understanding the mechanisms responsible for probiotic effects on the pathophysiology of NEC. Thousands of reports have been published in the past few years on the biological effects of various probiotics in various experimental systems.33 Experiments on intestinal epithelial cell function and intestinal pathophysiology have shown that these organisms improve the mucosal barrier, reduce mucosal permeability, decrease the pro-inflammatory response, increase the anti-inflammatory response, influence the colonization of pathogenic organisms, decrease TLR4 signaling, increase TLR9 (anti-inflammatory TLR response) signaling and many others (Figure 2).34 As many of these pathways are implicated in the pathogenesis of NEC, there is ample biological plausibility to explain probiotic effects on the developing intestine. Nonetheless, the specific mechanism(s) related to specific organisms in each situation remain poorly elucidated.
Although safety still remains a theoretical concern, in the trials reported above, there have been no cases of sepsis or other adverse events associated with the use of probiotics in premature infants. Nonetheless, there have been reports on probiotic sepsis in adults, children and infants, although primarily in immunocompromised patients. In one study, the supplemented probiotic was proven by DNA analysis to cause the subsequent blood infection. Although other adverse outcomes seem less likely, a recent adult study showed that a combination probiotic preparation increased the mortality rate in intensive care unit patients hospitalized with significant pancreatitis compared with that in controls.35 In addition, recent studies have shown an increased incidence of infection and wheezing in children who received probiotics during intensive care unit stays and through infancy.36 Although these trials require further scrutiny, widespread use of probiotics in high-risk, immunocompromised, premature infants might best be reserved until additional large multicenter trials have been completed.
Prebiotics are defined as undigested nutrients that influence intestinal microbial flora.37 Although human milk-fed infants have a ‘bifidogenic’ flora compared with formula-fed patients, studies have shown that human milk oligosaccharides contribute to this process. Human milk contains greater than 2 g l−1 oligosaccharides, and there are over 100 specific types of these molecules that appear to bind to specific organisms and actively provide host defense.38 Studies in full-term infants (and one in preterm infants) have shown that oligosaccharide supplementation into formula influences gut colonization similar to breast milk feeding toward a more bifidogenic flora.39 Furthermore, prebiotic supplementation has been shown to reduce atopic dermatitis and is associated with increased fecal IgA secretion in one particular report.40 These findings suggest that prebiotics may work similarly to probiotics and that there could be a rationale for use in preterm infants for the prevention of NEC. Nonetheless, prebiotics for NEC prevention have yet to be studied.
Should neonatologists jump on the probiotic bandwagon for the prevention of NEC?
Despite the real promise of probiotics for the prevention of NEC, several questions remain that preclude their use as a standard of care for preterm infants. First, there are several species with diverse effects, and the optimal preparation has not been clarified. In addition to the species, little is known regarding optimal dose, dosing strategy and whether live or attenuated probiotics are optimal for this condition. Furthermore, it is suggested that gut colonization with these organisms is important, yet no studies have confirmed that active colonization is necessary for disease prevention. Most importantly, additional information is needed to confirm that this approach is safe in this high-risk population. Although over 1000 preterm infants have been supplemented, the preparations have differed in the specificity of organism, and there is little federal regulation of probiotics when marketed as food additives. This federal regulation is needed as studies have shown that some preparations do not contain the active probiotics reported, and others have pathogenic organisms in the preparation. Finally, long-term effects of this approach should be evaluated, as these organisms can alter immune responses and microbial–epithelial cross talk, and therefore could result in many long-term effects. Nonetheless, probiotics are a promising approach for the prevention of neonatal NEC, and forthcoming studies may confirm the safety and efficacy, thereby supporting their routine use in neonatal intensive care units worldwide.
MS Caplan is a paid consultant for Sigma Tau Pharmaceuticals and a member of the Speakers Bureau for Mead Johnson Nutritionals and Ross Laboratories. This paper was based on a talk presented at the Evidence vs Experience in Neonatal Practices Fifth Annual CME Conference that was supported by an unrestricted educational grant from Dey, LP.
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Cite this article
Caplan, M. Probiotic and prebiotic supplementation for the prevention of neonatal necrotizing enterocolitis. J Perinatol 29, S2–S6 (2009). https://doi.org/10.1038/jp.2009.21
- necrotizing enterocolitis
- inflammatory necrosis
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