The Developing Microbiome of the Preterm Infant

The Developing Microbiome of the Preterm Infant

Clinical Therapeutics/Volume 38, Number 4, 2016 Review Article The Developing Microbiome of the Preterm Infant Mara E. DiBartolomeo, DO; and Erika C...

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Clinical Therapeutics/Volume 38, Number 4, 2016

Review Article

The Developing Microbiome of the Preterm Infant Mara E. DiBartolomeo, DO; and Erika C. Claud, MD The University of Chicago, Chicago, Illinois ABSTRACT Purpose: To determine the importance of the neonatal microbiome in intestinal and overall health. Method: A review of existing literature. Findings and Implications: The microbiome is increasingly understood to have a significant role in health and disease. However, the microbiome of the preterm infant is unique, with simple microbial communities exposed to a consistent diet in a regulated environment, and development from naive to stable under the influence of the neonatal intensive care unit. This early microbiome encounters a still developing host and thus has the potential to program fundamental pathways with implications for neonatal and later outcomes. (Clin Ther. 2016;38:733–739) & 2016 Elsevier HS Journals, Inc. All rights reserved. Key words: development, microbiome, necrotizing enterocolitis, preterm infant.

INTRODUCTION The microbiome represents all the bacteria living in or on the host. It has been termed “the forgotten organ,” with significant weight, genetic content, cellular content, and metabolic activity.1 It has been estimated that 100 trillion organisms, upward of 10 times the total number of cells in the human body, comprise the gut microbiome alone.2 The interaction between the intestine and its microbiome is a complex relation with risk and benefit for the host. In a healthy host, these microorganisms have a dramatic impact on intestinal health and gut function. Important research on the preterm infant microbiome has focused on the intestinal microbiome and neonatal Scan the QR Code with your phone to obtain FREE ACCESS to the articles featured in the Clinical Therapeutics topical updates or text GS2C65 to 64842. To scan QR Codes your phone must have a QR Code reader installed.

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necrotizing enterocolitis (NEC). However, the microbiome has the potential for impact beyond the local intestinal level. This review focuses both on the microbiome in NEC and on the potential for this initial microbiome to have broader health impact.

NEONATAL NEC NEC is a potentially fatal inflammatory bowel necrosis that primarily affects premature infants after the initiation of enteral feeding. On the basis of large, multicenter, neonatal network databases from the United States and Canada, the mean prevalence of the disorder is  7% among infants with a birth weight between 500 and 1500 g.3 For decades, NEC was the focus of various clinical and laboratory studies; however, NEC remains poorly understood, likely because its pathogenesis is multifactorial. The primary risk factors for NEC are prematurity, bacterial colonization, enteral feeding, and altered intestinal blood flow. One hypothesis that integrates these known risk factors is that intestinal injury in NEC may be the result of synergy in which enteral feeding results in colonization of the uniquely susceptible premature intestine with pathogenic bacteria, leading to an exaggerated inflammatory response.4 The preterm infant is essentially a fetus, expecting the conditions of the intrauterine environment. For the intestine this includes limited contact with bacteria and food substrate. Microbial colonization of the still immature intestine occurs in the context of an incompletely developed innate immune defense system. This includes altered intestinal mucus, decreased barrier function, decreased levels of protective factors from goblet cells such as trefoil factor, and decreased paneth

Accepted for publication February 3, 2016. 0149-2918/$ - see front matter & 2016 Elsevier HS Journals, Inc. All rights reserved.


Clinical Therapeutics cell-derived defensins.5 These immaturities increase contact of the preterm gut with the colonizing bacteria. The exaggerated inflammatory response of the immature intestine to both commensal and pathogenic bacteria then leads to excessive inflammation with resultant ischemia and intestinal necrosis, causing the signs and symptoms of disease.6 NEC classically occurs between 7 and 14 days of life, but susceptibility is inversely related to gestational age. In very low birth weight (VLBW) infants it can occur up to several weeks of life with a peak incidence around 32 corrected weeks’ gestation, emphasizing the role of intestinal development in this process.7 There is no sex or race predilection. Disease presentation can range from subtle and nonspecific gastrointestinal (GI) signs of feeding intolerance, increased gastric residuals, and increased abdominal girth to fulminant signs/ symptoms of systemic shock associated with bowel ischemia and perforation.3 Pneumatosis intestinalis is the pathognomonic sign for NEC seen on radiographs. It represents air from bacterial fermentation of intraluminal food substrate tracking within the bowel wall. The most severe cases are necrosis of the entire intestine and are termed NEC totalis.8 Because pathogenesis of this disease is unknown, treatment is nonspecific. Current treatment strategies are aimed at supportive care of the infant, including bowel rest, antibiotic administration, nasogastric tube placement for decompression, addressing acid/base disturbances, and following closely for clinical and radiographic signs of intestinal perforation. Perforation or clinical deterioration associated with ongoing intestinal necrosis is indication for surgical intervention by either peritoneal drain placement or exploratory laparotomy.9 Current treatment methods are often inadequate because of the rapid progression of NEC from its initial diagnosis. There is a high mortality rate of 20% to 30%, up to 50% in infants who require surgery.3 Morbidity includes risk of stricture, short-gut, and intestinal failure. Furthermore, studies have reported that NEC, particularly surgical NEC, is an independent risk factor for poor neurodevelopmental outcome.10 The pathophysiologic cascade leading to NEC once started is difficult to stop, suggesting that understanding early aspects of the intestinal injury that precede clinical signs and strategies for prevention are of paramount importance. This has led to a specific interest in understanding the role of the preterm infant microbiome in injury and protection from NEC.


THE MICROBIOME AND NEC The microbiome is an ecologic system. A healthy ecosystem is characterized by a high diversity of species with accompanying balance, functional redundancy, and resistance to disease. In contrast, a sick ecosystem is termed dysbiosis and is characterized by a low diversity of species, imbalance, and lack of functional redundancy with resultant susceptibility to disease. Diversity in preterm infants decreases throughout their neonatal intensive care unit (NICU) stay, and NEC is associated with dysbiosis.11 We have previously used 16s rRNA sequencing techniques to examine the microbiome of patients who develop NEC compared with healthy controls.12 We found that the microbiome of preterm infants with NEC shifts up to 3 weeks before disease onset with a decrease in Firmicutes and an increase in Proteobacteria.13 These data suggest a window of opportunity for intervention to prevent pathogenic shift. Additional studies have examined the development of the healthy preterm infant microbiome over time. The microbiome was again examined by 16s rRNA sequencing of prospectively collected fecal samples over the first 8 weeks of life. Preterm infants without NEC were compared with a vaginally delivered, breastfed, full-term infant, cared for at home. The weekly samples for the full-term infant over the first 8 weeks of life clustered closely together at all time points.13 In contrast, healthy premature infants exhibited clustering at o2 weeks of age, 3 to 5 weeks of age, and 46 weeks of age.13 This study indicates that timing of microbiome development is important. Together these studies suggest that understanding the early microbiome is key for understanding normal microbiome development in patients who remain healthy without NEC and for understanding what alterations lead to NEC.

THE MICROBIOME AND OUTCOMES The microbiome of the preterm infant is unique, with simple microbial communities, exposure to a consistent diet in a regulated environment, and development in the context of a simultaneously developing host. A premature infant is vastly different from a full-term infant, with many different needs. It is not just size. Full-term infants have completed in utero development, whereas preterm infants have not. This includes development of all organ systems and of the microbiome itself. The “core” needs of the preterm

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M.E. DiBartolomeo and E.C. Claud infant may be fundamentally different from those of a full-term infant, and the “core” microbiota for health particularly in the early stages may also be unique. Various factors influence the composition of the premature intestinal microbiome over time. Recent data suggest that colonization of the GI tract may begin in utero.14 Vertical transmission of maternal microbiota is thought to occur before and during the birthing process, with studies reporting a microbiome within fetal meconium and maternal placenta.15,16 Mode of delivery influences the newborn microbiome as well, with the microbiome of newborns delivered by cesarean delivery characterized by a lack of strict anaerobes and the presence of facultative anaerobes such as Clostridium species.17–19 The NICU also influences the microbiome of preterm infants. This environment includes clinical interventions such as instrumentation, exposure to antibiotics and histamine H2-receptor antagonist blockers, and alterations in diet, such as formula feeding, breast milk fortifier additions, and periods of fasting.20–22 The microbiome of the preterm infant must support normal intestinal and immune system development and remain resistant and resilient to NICU stress. We have examined the function of preterm infant microbial communities with a humanized gnotobiotic mouse model. To investigate the effect of early (o2 weeks of life) preterm human infant microbiota, gnotobiotic pregnant dams were gavaged with the infant microbiota of interest.23 Resulting pups acquired this microbiota of interest naturally through birth and nursing. In preterm infants, growth can be used as a surrogate for health.24 We thus specifically chose early human fecal samples from 2 preterm infants of the same gestational age and yet with persistent differences in their growth rate from birth until discharge. The microbiome from the infant with poor growth induced a significant upregulation of genes and pathways associated with innate immune/inflammatory responses in the transfaunated pups.23 In contrast, despite a similar gut microbiota at the phylum level, the microbiome from the infant with normal growth displayed a downregulation of these inflammation-associated genes, suggesting the existence of a microbial community that may protect certain preterm infants.23 The increased inflammatory gene expression in pups transfaunated with microbiota from the poor growth infant was associated with increased baseline serum inflammatory cytokine levels even without a secondary

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inflammatory insult.23 Because much of the morbidity of prematurity is inflammatory in nature, an increased inflammatory profile could alter outcomes in addition to NEC incidence.

MICROBIOTA-GUT-BRAIN AXIS Studies in animals have found that systemic inflammatory insults in early life can lead to increased proliferation of microglia within the hippocampus and inhibition of neurogenesis and neuronal development.25,26 Thus, altered microbial communities could influence neurodevelopmental outcomes. Neurodevelopmental outcomes are of prime importance to neonatology. Brain immaturity makes the preterm infant more vulnerable to inflammatory insult. It is thought that neurogenesis and neuronal maturation play a large role in overall brain maturation well into the second and third trimesters of pregnancy, which makes infants born prematurely, especially VLBW infants, a vulnerable population.27 A direct “microbiota-gut-brain” connection exists via bidirectional neural and immune interactions of the intestine and the brain.28 The GI tract is home to millions of neurons and the 100 trillion bacteria that comprise the human gut microbiota, setting the stage for this connection.29 In a “healthy setting,” with “normal” gut microbiota there is both healthy central nervous system and healthy gut function, with normal behavior, cognition, and emotion, and a healthy level of inflammatory cells. In contrast, dysbiosis, in the setting of stress and disease, has been associated with alterations in behavior, cognition, emotion, and altered levels of inflammatory cells.1 The brain can influence GI functions such as motility, whereas the gut can influence neural responses such as stress.1 Germ-free mice have altered risk-taking behavior, memory, and anxiety.1,30 In addition, animal models that were designed for neurobehavioral phenotypes have unexpectedly found GI disease such as increased GI permeability, altered tight junction protein expression, and abnormal intestinal cytokine profiles.31 Clinically, GI complaints and increased incidence of inflammatory bowel disease are commonly reported with autism spectrum disorder.32,33 One study that compared the intestinal microbiome of patients with autism spectrum disorder with controls found alterations specifically in Clostridia, Bacteroidetes, and Desulfovibrio.34 Further study


Clinical Therapeutics revealed increased levels of propionic acid, a short-chain fatty acid fermentation product of Clostridia, Bacteroidetes, and Desulfovibrio.34 Tests in animal models found that propionic acid induces neurochemical changes in rat brain tissue associated with abnormal motor movements, electrographic changes, and impaired social interactions that are characteristic of autism.34 NEC is known to be associated with significantly worse neurodevelopmental outcomes than prematurity alone.10 This is likely multifactorial. When VLBW infants are affected by NEC, there is a perfect storm for both prematurity-related delays in brain maturation and damage to existing cerebral tissue, because these infants are in extremis compared with similar cohorts of VLBW infants. Infants affected by NEC can be extremely ill, on pharmacologic blood pressure support to maintain end-organ perfusion, and on longstanding total parenteral nutrition. They may be acidotic and have hypoglycemia or hyperglycemia. Severe NEC requires surgical intervention. Correspondingly, it is known that nutrition affects postnatal brain maturation.28 Illness, with concurrent infection, inflammation, and acidosis, affects neurogenesis and synaptogenesis.35 Inflammation alone was found to increase the risk of VLBW infants developing periventricular leukomalacia and associated poor neurodevelopmental outcomes.36 Exposures to anesthesia and sedation were also found to affect brain maturation and neurodevelopmental outcomes.37 Dysbiosis associated with NEC may also contribute to the poor neurodevelopmental outcome of these infants.

OPTIMIZING THE PRETERM INFANT MICROBIOME The microbiome has been associated with NEC, growth, neurodevelopmental outcome, inflammatory bowel disease, obesity, autism, asthma, and celiac disease.12,38–43 The early microbiome thus has the potential to alter fundamental processes and to affect outcomes beyond the neonatal period. Recognition of this importance has led to an interest in strategies to optimize the microbiome. Specifically for the preterm infant, studies have found a temporal clustering at o2 weeks of life; thus, there is interest in strategies to specifically influence this early microbiome.13 One can manipulate the early microbiome with interventions such as probiotics or can protect the microbiome via


strategies such as limiting empiric antibiotics and increasing human milk (HM) feeding. Most probiotics used in preterm infants are designed to correct the dysbiosis of prematurity and to shift the preterm infant microbiome toward that of a “healthy” full-term infant.44–47 The impact of probiotics specifically on NEC has become one of the most studied interventions in neonatal medicine. A key meta-analysis performed in 2010 titled “Probiotics for Prevention of Necrotizing Enterocolitis in Preterm Infants” reviewed 24 randomized trials and 45000 preterm infants.44 Probiotics administered via multiple protocols and with different probiotic agents were combined, although a common feature was administering probiotics early in life, near first feed. This meta-analysis concluded that enteral administration of probiotics reduces the incidence of severe NEC, mortality and NEC-related mortality.44 Possible mechanisms of action include an increased barrier to the migration of bacteria and their products across the intestinal mucosa,48 competitive exclusion of potential pathogens,49 modification of host response to microbial products,50 augmentation of immunoglobulin A mucosal responses, and upregulation of immune responses.51 Cautions for use of probiotics in preterm infants include lack of regulatory control of probiotic products, case reports of probiotic-associated sepsis and associated fatalities, and poor understanding of their effect on the developing microbiome.52 Although NEC is a terrible disease, most infants do not develop NEC. Administering probiotics to all preterm infants alters the microbiome at key early time points in unknown ways for many who were never going to develop NEC. There are alternative means of influencing the initial microbiome. Diet is known to influence the microbiome. A study by Meinzen-Derr et al53 analyzed the HM intake of 1272 VLBW infants. HM intake was defined as the proportion of own mother’s milk to total intake over the first 14 days.53 This study found that HM in the first 14 days of life decreases the risk of NEC in a dose-dependent manner. This is the specific early time window found to have distinct clustering (o2 weeks of life). The impact of antibiotic administration on NEC was also studied, again, with a focus on alteration of the neonatal microbiome. Given concerns for underlying infections in the setting of preterm deliveries, concern for early-onset sepsis in premature infants and the overall vulnerability of VLBW infants, antibiotics

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M.E. DiBartolomeo and E.C. Claud are frequently started empirically in NICUs.54 Antibiotics are the most commonly prescribed medications in NICUs.55 Administration of antibiotics and overall antibiotic exposure were found to perturb the neonatal microbiome and to decrease fecal microbial diversity.56 In a retrospective cohort analysis of VLBW infants, Cotten et al57 evaluated patients who had received initial empirical antibiotic therapy and had sterile culture results in the first 3 days of life. This study defined prolonged empirical antibiotic therapy as Z5 days and found that prolonged therapy was associated with increased odds of NEC or death. Specifically, a 7% increase in the odds of NEC was observed for each additional day of initial empirical antibiotic treatment.57 This study, again, found the impact of a clinical intervention that alters the microbiome at o2 weeks of life. This impact of antibiotics on NEC incidence is consistent with findings in microbiome-based studies. When 16s rRNA sequencing was used for analysis of fecal samples from 20 preterm infants, 10 with NEC and 10 matched controls (including 4 twin pairs) from a single-site level III NICU, it was found that patients with NEC had microbial communities that clustered separate from those of controls.12 Patients with NEC had lower diversity than controls and a bloom of Gammaproteobacteria. The only significant clinical difference between groups was number of days of antibiotics before disease onset, with those developing NEC having significantly more days of previous antibiotics.12

IMPLICATIONS The microbiome is important in health and disease. The infant microbiome, specifically the preterm infant microbiome, has the potential to influence disease development during infancy (NEC, growth, neurodevelopmental outcome) and later health outcomes (obesity, autism, allergy). Thus, efforts to manipulate the microbiome must be used with caution. Although administration of probiotics is appealing, the term “probiotics” encompasses many different organisms, and the effect of probiotics on the preterm infant microbiome is unknown. However, there are other means of optimizing the microbiome and decreasing NEC incidence that protect the developing preterm infant microbiome. This includes decreasing empiric

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antibiotic use and promoting exclusive own mother’s milk feeding. Clinicians must recognize that the microbiome is influenced by all NICU interventions. Therapeutic options should be considered in light of effect on the microbiome. In infancy when health begins, one has a great opportunity to establish or compromise beneficial microbial patterns for a healthy lifetime.

ACKNOWLEDGMENTS This work was supported by the National Institute of Child Health and Human Development grant R01 HD 083481 (ECC). Both authors contributed to the literature search and writing of this manuscript.

CONFLICTS OF INTEREST There are no study sponsors or conflicts of interest.

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31. Hsiao EY, McBride SW, Hsien S, et al. Microbiota modulate behavioral and physiological abnormalities associated with neurodevelopmental disorders. Cell. 2013;155: 1451–1463. 32. Doshi-Velez F, Avillach P, Palmer N, et al. Prevalence of inflammatory bowel disease among patients with autism spectrum disorders. Inflamm Bowel Dis. 2015;21:2281–2288. 33. Parracho HM, Bingham MO, Gibson GR, McCartney AL. Differences between the gut microflora of children with autistic spectrum disorders and that of healthy children. J Med Microbiol. 2005;54:987–991. 34. Macfabe DF. Short-chain fatty acid fermentation products of the gut microbiome: implications in autism spectrum disorders. Microb Ecol Health Dis. 2012:23. 35. Rees CM, Pierro A, Eaton S. Neurodevelopmental outcomes of neonates with medically and surgically treated necrotizing enterocolitis. Arch Dis Child Fetal Neonatal Ed. 2007;92:F193–F198. 36. Ellison VJ, Mocatta TJ, Winterbourn CC, et al. The relationship of CSF and plasma cytokine levels to cerebral white matter injury in the premature newborn. Pediatr Res. 2005;57:282–286. 37. Sun L. Early childhood general anaesthesia exposure and neurocognitive development. Br J Anaesth. 2010;105(Suppl 1):i61–i68. 38. Ismail IH, Oppedisano F, Joseph SJ, et al. Reduced gut microbial diversity in early life is associated with later development of eczema but not atopy in high-risk infants. Pediatr Allergy Immunol. 2012;23:674– 681. 39. Kronman MP, Zaoutis TE, Haynes K, et al. Antibiotic exposure and IBD development among children: a population-based cohort study. Pediatrics. 2012;130:e794–e803. 40. Penders J, Thijs C, van den Brandt PA, et al. Gut microbiota composition and development of atopic

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manifestations in infancy: the KOALA Birth Cohort Study. Gut. 2007;56:661–667. Saari A, Virta LJ, Sankilampi U, et al. Antibiotic exposure in infancy and risk of being overweight in the first 24 months of life. Pediatrics. 2015;135:617–626. Mai V, Young CM, Ukhanova M, et al. Fecal microbiota in premature infants prior to necrotizing enterocolitis. PLoS One. 2011;6:e20647. Petrof EO, Claud EC, Gloor GB, Allen-Vercoe E. Microbial ecosystems therapeutics: a new paradigm in medicine? Benef Microbes. 2013;4: 53–65. AlFaleh K, Anabrees J. Probiotics for prevention of necrotizing enterocolitis in preterm infants. Cochrane Database Syst Rev. 2014;4:CD005496. Bin-Nun A, Bromiker R, Wilschanski M, et al. Oral probiotics prevent necrotizing enterocolitis in very low birth weight neonates. J Pediatr. 2005;147:192–196. Lin HC, Hsu CH, Chen HL, et al. Oral probiotics prevent necrotizing enterocolitis in very low birth weight preterm infants: a multicenter, randomized, controlled trial. Pediatrics. 2008;122:693–700. Lin HC, Su BH, Chen AC, et al. Oral probiotics reduce the incidence and severity of necrotizing enterocolitis in very low birth weight infants. Pediatrics. 2005;115:1–4. Mattar AF, Drongowski RA, Coran AG, Harmon CM. Effect of probiotics on enterocyte bacterial translocation in vitro. Pediatr Surg Int. 2001;17:265–268. Reid G, Howard J, Gan BS. Can bacterial interference prevent infection? Trends Microbiol. 2001;9:424– 428. Duffy LC. Interactions mediating bacterial translocation in the immature intestine. J Nutr. 2000;130: 432S–436SS. Link-Amster H, Rochat F, Saudan KY, et al. Modulation of a specific humoral immune response and

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Address correspondence to: Erika C. Claud, MD, The University of Chicago, 5841 S. Maryland Ave MC6060, Chicago, IL 60637. E-mail: [email protected]